foundation repair contractor Vista

Shallow foundation

July 13, 2017 update: For the latest, please read “Zebras: Let’s Get In Formation.”A year ago we wrote “Sex & Startups.” The premise was this: The current technology and venture capital structure is broken. It rewards quantity over quality, consumption over creation, quick exits over sustainable growth, and shareholder profit over shared prosperity. It chases after “unicorn” companies bent on “disruption” rather than supporting businesses that repair, cultivate, and connect. After publishing the essay, we heard from hundreds of founders, investors, and advocates who agreed: “We cannot win at this game.”This is an urgent problem. For in this game, far more than money is at stake. When VC firms prize time on site over truth, a lucky few may profit, but civil society suffers. When shareholder return trumps collective well-being, democracy itself is threatened. The reality is that business models breed behavior, and at scale, that behavior can lead to far-reaching, sometimes destructive outcomes.Facebook — the ultimate unicorn — was weaponized to spread fake news during the presidential election. Uber has come under fire for supporting dubious political agendas, tolerating a toxic workplace culture, manipulating employee wages, and circumnavigating regulations. Medium has backpedaled, having realized that while clickbait content may produce the ad-revenue hockey stick investors want to see, it undermines the founders’ original mission to create a publishing model that enlightens, informs, and rewards quality over quantity.Many well-reasoned think pieces* have been written about the gaping chasm between the world we need and the world that exists. Today, we want to provide the seeds of a solution — and to encourage founders, investors, foundations, corporations, and their allies to organize around it.A company’s business model is the first domino in a long chain of consequences. In short: “The business model is the message.” From that business model flows company culture and beliefs, strategies for success, end-user experiences, and, ultimately, the very shape of society.We believe that developing alternative business models to the startup status quo has become a central moral challenge of our time. These alternative models will balance profit and purpose, champion democracy, and put a premium on sharing power and resources. Companies that create a more just and responsible society will hear, help, and heal the customers and communities they serve.Think of our most valuable institutions — journalism, education, healthcare, government, the “third sector” of nonprofits and social enterprises — as houses upon which democracy rests. Unicorn companies are rewarded for disrupting these, for razing them to the ground.Instead, we ought to support companies that provide extreme home makeovers. We can’t assume these companies will be created by accident. We must intentionally build the infrastructure to nurture them.ENTER THE ZEBRAThis new movement demands a new symbol, so we’re claiming an animal of our own: the zebra.Why zebras?To state the obvious: unlike unicorns, zebras are real.Zebra companies are both black and white: they are profitable and improve society. They won’t sacrifice one for the other.Zebras are also mutualistic: by banding together in groups, they protect and preserve one another. Their individual input results in stronger collective output.Zebra companies are built with peerless stamina and capital efficiency, as long as conditions allow them to survive.The capital system is failing society in part because it is failing zebra companies: profitable businesses that solve real, meaningful problems and in the process repair existing social systems.Drawing from the work of many thinkers,** we’ve developed a portrait of what a zebra company is, does, and stands for. This chart outlines how a zebra company compares with its mythical cousin, the unicorn.For a downloadable, printable version, click here.WHY IS IT SO HARD TO BUILD ZEBRA COMPANIES?In the last year we’ve spoken to countless founders, investors, foundations, and thought leaders who believe zebra companies are crucial to our society’s success. Yet zebras struggle for survival because they lack the environment to encourage their birth, let alone to support them through maturity. “I wonder how many change-makers are stuck under the demands of unicorn investors,” said TJ Abood of Access Ventures, who added that he worried about “the opportunity cost to society” under this model.From our conversations with stakeholders, we distilled the most common challenges facing zebra companies:1. The problem isn’t product, it’s process. Tech isn’t a silver bullet. Building more won’t solve the biggest challenges we face today. An app won’t address the homelessness crisis in San Francisco or unite bitterly divided partisan politicians. The obstacle is that we are not investing in the process and time it takes to help institutions adopt, deploy, and measure the success of innovation, apps or otherwise.2. Zebra companies are often started by women and other underrepresented founders. Three percent of venture funding goes to women and less than one percent to people of color. Although women start 30 percent of businesses, they receive only 5 percent of small-business loans and 3 percent of venture capital. Yet when surveyed, women — who perform better overall than founding teams composed exclusively of men — say they are in it for the long haul: to build profitable, sustainable companies.3. You can’t be it if you can’t see it. Look hard outside of Silicon Valley and you’ll find promising zebra companies. But existing and aspiring business owners haven’t seen enough proof that they’ll have a higher chance of becoming financially successful and socially celebrated if they follow sustainable business practices. They lack heroes to emulate, so they default to the “growth at all costs” model. Imagine if every fund allocated a small percentage for zebra experiments. The investing firm has bravely stepped into this space, but it shouldn’t stand alone.4. Zebras are stuck between two outdated paradigms, nonprofit and for-profit. For young companies pursuing both profit and purpose, the existing imperfect structures (hybrid for-profit/nonprofit, Public Benefit Corps, B-Corps, L3Cs) can be prohibitively expensive. The expense comes not only in legal fees, but in the consumption of a founder’s most precious commodity: time. Months are lost searching for aligned, strategic investors who are both familiar and comfortable with alternative models. This presents a chicken-and-egg problem for foundations, philanthropists, and investors alike. They are spooked by unproven alternative models, but companies can’t prove their models work without experiments to fund them in the first place. Moreover, the current tax system doesn’t reward — or even acknowledge — anything other than for-profit (tax) or nonprofit (deduction) strategies. From the IRS’s perspective, there is nothing akin to a “50 percent financial return, 50 percent social impact” investment. This leaves many potential investors in a straitjacket.5. Impact investing’s thesis is detrimentally narrow and risk-averse. Much of the $36 billion in impact investment funding is restricted to verticals like clean technology, microfinance, or global health. This immature market limits innovation in other sectors — like journalism and education — that could desperately use it.“So how will investors turn a profit and mitigate risks?” you may be asking. Dividends? Equity crowdfunding? We don’t have all the answers. But we’ve seen how a company’s business model and values can negatively affect the bottom line (#deleteuber). So what if the opposite is also true? What if more-enlightened dollars invested in more-enlightened companies led to stronger returns? What if companies that stood for something were in fact more profitable? Patagonia, Warby Parker, Zingerman’s, Etsy, Mailchimp, Basecamp, and Kickstarter are a start — but the world needs so much more.MAKE ZEBRAS: JOIN USIf you believe technology and capital must do better, if you are building a zebra company or want to help carve out a space for them to thrive: join usOur goal is to gather zebra founders, philanthropists, investors, thinkers, and advocates to meet in person this year for DazzleCon (November 15–17 in Portland, Oregon)— a group of zebras is called a dazzle! — to learn from one another and pool resources, ideas, and best practices, to collectively advance this set of ambitions. From this gathering, we will capture and share the unique patterns that zebra founders and funders are finding, and we’ll turn a loose network into a powerful, cohesive movement.Are you in? Go here.Jennifer Brandel is the co-founder and CEO of Hearken. Mara Zepeda is the co-founder and CEO of Switchboard. Extra special thanks to Astrid Scholz, founder and CEO of Sphaera.DazzleCon is proudly supported by Knight Foundation, Artha Investing for Impact, Social Capital Markets, Portland Incubator Experiment, and Catalyst Law. Interested in sponsoring? Get in touch.*Thought piece roundup: Anil Dash on Humane Tech, Joe Edelman’s “Nothing to be Done,” Victoria Fram’s “Why An Equity-Only Investment Strategy Overlooks Many Promising Entrepreneurs,” Caterina Fake’s “The Age of the Cockaroach,” Jason Fried’s “Why We Chose Profit,” Christie George’s “Investing in Sharing,” Tristan Harris’s “How Technology Hijacks People’s Minds — from a Magician and Google’s Design Ethicist,” David Heinemeier Hansson’s “Exponential Growth Devours and Corrupts,” Julie Menter’s “On Unicorns: We’re Looking for a Different Kind of Magic,” Tim O’Reilly’s “We’ve Got This Whole Unicorn Thing Wrong,” Andrew Wilkinson’s “Unicorns vs. Horses.”**Jenn Armbrust’s Feminist Business School, Amber Case’s Calm Technology, Jerry Colonna, Ali Schultz and the work of, Anil Dash in conversation with Krista Tippett, in On Being; Seth Godin’s Tribes: We Need You to Lead Us; Jonathan Harris’ Modern Medicine; Ivan Illich’s Deschooling Society; Michael Karlberg’s “Beyond the Culture of Contest”; Jerry Michalski’s “What if we Trusted You?,” Howard Rheingold’s “Toward a Literacy of Cooperation”; Douglas Rushkoff’s Throwing Rocks at the Google Bus; Natasha Dow Schüll’s Addiction by Design; Shel Silverstein’s poem “Zebra Question”; Peter Thiel’s Zero to One; Ari Weinzweig’s Anarchist’s Guide to Building a Better Business series. Zebra companies have been referred to in journalism articles and academic papers, and by some VC firms.Thanks to Jenn Armbrust, Adam Brault, David Chen, Molly deAguiar, John Dimatos, Lennon Day-Reynolds, Corey Ford, Christie George, Seth Godin, Andrew Haeg, Jason Kunesh, Rachel Hankerson, Jonathan Harris, Jennifer Jordan, Luke Kanies, Duncan Malashock, Kanyi Maqubela, Ellen Mayer, Julie Menter, Douglas Rushkoff, Jake Shapiro, Michael Slaby, Rick Turoczy, Stephanie Pereira, April Rinne, Tom Watson. And to Jen McDonald for editing; and to Arthur Jones for the illustrations.


DNA damage resulting in multiple broken chromosomes DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.[1] Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages (interstrand crosslinks or ICLs).[2][3] This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states: an irreversible state of dormancy, known as senescence cell suicide, also known as apoptosis or programmed cell death unregulated cell division, which can lead to the formation of a tumor that is cancerous The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA damage repair and protection.[4] Play media Paul Modrich The 2015 Nobel Prize in Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar for their work on the molecular mechanisms of DNA repair processes.[5][6] There are two types: nucleotide excision repair and base excision repair. Further information: DNA damage (naturally occurring) and Free radical damage to DNA DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.[1] While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation and contribute to tumour heterogeneity. The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage. DNA damage can be subdivided into two main types: endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination also includes replication errors exogenous damage caused by external agents such as ultraviolet [UV 200–400 nm] radiation from the sun other radiation frequencies, including x-rays and gamma rays hydrolysis or thermal disruption certain plant toxins human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents viruses[7] The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for example, through gene conversion). There are several types of damage to DNA due to endogenous cellular processes: oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species, alkylation of bases (usually methylation), such as formation of 7-methylguanosine, 1-methyladenine, 6-O-Methylguanine hydrolysis of bases, such as deamination, depurination, and depyrimidination. "bulky adduct formation" (i.e., benzo[a]pyrene diol epoxide-dG adduct, aristolactam I-dA adduct) mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted. Monoadduct damage cause by change in single nitrogenous base of DNA Diadduct damage Damage caused by exogenous agents comes in many forms. Some examples are: UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands. Intermediate-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional errors needed for neoplasia or may trigger viral interactions) leading to pre-mature aging and cancer. Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA backbone) and single-strand breaks. For example, hydrolytic depurination is seen in the thermophilic bacteria, which grow in hot springs at 40–80 °C.[8][9] The rate of depurination (300 purine residues per genome per generation) is too high in these species to be repaired by normal repair machinery, hence a possibility of an adaptive response cannot be ruled out. Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and crosslinking of DNA, just to name a few. UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift. In human cells, and eukaryotic cells in general, DNA is found in two cellular locations — inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells. Senescence, an irreversible process in which the cell no longer divides, is a protective response to the shortening of the chromosome ends. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (see Hayflick limit).[10] In contrast, quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the organism,[11] which serves as a "last resort" mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis is considered to be part of a strategy of protection against cancer.[12] It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damage and mutation are fundamentally different. Damage results in physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damage can be recognized by enzymes, and thus can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and thus translation into a protein will also be blocked. Replication may also be blocked or the cell may die. In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and thus a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce. Although distinctly different from each other, DNA damage and mutation are related because DNA damage often causes errors of DNA synthesis during replication or repair; these errors are a major source of mutation. Given these properties of DNA damage and mutation, it can be seen that DNA damage is a special problem in non-dividing or slowly-dividing cells, where unrepaired damage will tend to accumulate over time. On the other hand, in rapidly-dividing cells, unrepaired DNA damage that does not kill the cell by blocking replication will tend to cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are deleterious to a cell's survival. Thus, in a population of cells composing a tissue with replicating cells, mutant cells will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism, because such mutant cells can give rise to cancer. Thus, DNA damage in frequently dividing cells, because it gives rise to mutations, is a prominent cause of cancer. In contrast, DNA damage in infrequently-dividing cells is likely a prominent cause of aging.[13] Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when non-essential genes are missing or damaged). Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort. Damage to DNA alters the spatial configuration of the helix, and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place. Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can occur in only one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300–500 nm wavelength) to promote catalysis.[14] Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans,[15] who instead use nucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used only once; that is, the reaction is stoichiometric rather than catalytic.[16] A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes.[17] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine. Structure of the base-excision repair enzyme uracil-DNA glycosylase excising a hydrolytically-produced uracil residue from DNA. The uracil residue is shown in yellow. When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.[16] Base excision repair (BER) repairs damage to a single nitrogenous base by deploying enzymes called glycosylases.[18] These enzymes remove a single nitrogenous base to create an apurinic or apyrimidinic site (AP site).[18] Enzymes called AP endonucleases nick the damaged DNA backbone at the AP site. DNA polymerase then removes the damaged region using its 5’ to 3’ exonuclease activity and correctly synthesizes the new strand using the complementary strand as a template.[18] Nucleotide excision repair (NER) repairs damaged DNA which commonly consists of bulky, helix-distorting damage, such as pyrimidine dimerization caused by UV light. Damaged regions are removed in 12–24 nucleotide-long strands in a three-step process which consists of recognition of damage, excision of damaged DNA both upstream and downstream of damage by endonucleases, and resynthesis of removed DNA region.[19] NER is a highly evolutionarily conserved repair mechanism and is used in nearly all eukaryotic and prokaryotic cells.[19] In prokaryotes, NER is mediated by Uvr proteins.[19] In eukaryotes, many more proteins are involved, although the general strategy is the same.[19] Mismatch repair systems are present in essentially all cells to correct errors that are not corrected by proofreading. These systems consist of at least two proteins. One detects the mismatch, and the other recruits an endonuclease that cleaves the newly synthesized DNA strand close to the region of damage. In E. coli , the proteins involved are the Mut class proteins. This is followed by removal of damaged region by an exonuclease, resynthesis by DNA polymerase, and nick sealing by DNA ligase.[20] Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. PVN Acharya noted that double-strand breaks and a "cross-linkage joining both strands at the same point is irreparable because neither strand can then serve as a template for repair. The cell will die in the next mitosis or in some rare instances, mutate."[2][3] Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination.[16][21] In an in vitro system, MMEJ occurred in mammalian cells at the levels of 10–20% of HR when both HR and NHEJ mechanisms were also available.[22] DNA ligase, shown above repairing chromosomal damage, is an enzyme that joins broken nucleotides together by catalyzing the formation of an internucleotide ester bond between the phosphate backbone and the deoxyribose nucleotides. In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends.[23] To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate.[24][25][26][27] NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are "backup" NHEJ pathways in higher eukaryotes.[28] Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.[29] Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination. MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions.[22] In further steps,[30] Poly (ADP-ribose) polymerase 1 (PARP1) is required and may be an early step in MMEJ. There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. This is followed by recruitment of XRCC1–LIG3 to the site for ligating the DNA ends, leading to an intact DNA. MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.[31] The extremophile Deinococcus radiodurans has a remarkable ability to survive DNA damage from ionizing radiation and other sources. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.[32] Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them. Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.[33] It involves switching out regular DNA polymerases for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol η mediates error-free bypass of lesions induced by UV irradiation, whereas Pol ι introduces mutations at these sites. Pol η is known to add the first adenine across the T^T photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or repairing lesions at locations of stalled DNA replication. For example, Human DNA polymerase eta can bypass complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although can cause targeted and semi-targeted mutations.[34] Paromita Raychaudhury and Ashis Basu[35] studied the toxicity and mutagenesis of the same lesion in Escherichia coli by replicating a G[8,5-Me]T-modified plasmid in E. coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication.[36][37] After translesion synthesis, extension is required. This extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol η, yet if TLS results in a mismatch, a specialized polymerase is needed to extend it; Pol ζ. Pol ζ is unique in that it can extend terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol ι to fix the lesion, then PCNA may switch to Pol ζ to extend the mismatch, and last PCNA will switch to the processive polymerase to continue replication. Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage.[38] The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division. The packaging of eukaryotic DNA into chromatin presents a barrier to all DNA-based processes that require recruitment of enzymes to their sites of action. To allow DNA repair, the chromatin must be remodeled. In eukaryotes, ATP dependent chromatin remodeling complexes and histone-modifying enzymes are two predominant factors employed to accomplish this remodeling process.[39] Chromatin relaxation occurs rapidly at the site of a DNA damage.[40] In one of the earliest steps, the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to double-strand breaks or other DNA damage.[41] This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites, and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs.[41] PARP1 protein starts to appear at DNA damage sites in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs.[42] PARP1 synthesizes polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chains on itself. Next the chromatin remodeler ALC1 quickly attaches to the product of PARP1 action, a poly-ADP ribose chain, and ALC1 completes arrival at the DNA damage within 10 seconds of the occurrence of the damage.[40] About half of the maximum chromatin relaxation, presumably due to action of ALC1, occurs by 10 seconds.[40] This then allows recruitment of the DNA repair enzyme MRE11, to initiate DNA repair, within 13 seconds.[42] γH2AX, the phosphorylated form of H2AX is also involved in the early steps leading to chromatin decondensation after DNA double-strand breaks. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin.[43] γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute.[43] The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break.[43] γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX.[44] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4,[45] a component of the nucleosome remodeling and deacetylase complex NuRD. DDB2 occurs in a heterodimeric complex with DDB1. This complex further complexes with the ubiquitin ligase protein CUL4A[46] and with PARP1.[47] This larger complex rapidly associates with UV-induced damage within chromatin, with half-maximum association completed in 40 seconds.[46] The PARP1 protein, attached to both DDB1 and DDB2, then PARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein ALC1.[47] Action of ALC1 relaxes the chromatin at the site of UV damage to DNA. This relaxation allows other proteins in the nucleotide excision repair pathway to enter the chromatin and repair UV-induced cyclobutane pyrimidine dimer damages. After rapid chromatin remodeling, cell cycle checkpoints are activated to allow DNA repair to occur before the cell cycle progresses. First, two kinases, ATM and ATR are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint protein Chk1, initiating its function, about 10 minutes after DNA is damaged.[48] After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,[49] whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified.[50] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins. DNA damage checkpoint is a signal transduction pathway that blocks cell cycle progression in G1, G2 and metaphase and slows down the rate of S phase progression when DNA is damaged. It leads to a pause in cell cycle allowing the cell time to repair the damage before continuing to divide. Checkpoint Proteins can be separated into four groups: phosphatidylinositol 3-kinase (PI3K)-like protein kinase, proliferating cell nuclear antigen (PCNA)-like group, two serine/threonine(S/T) kinases and their adaptors. Central to all DNA damage induced checkpoints responses is a pair of large protein kinases belonging to the first group of PI3K-like protein kinases-the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia- and Rad-related) kinases, whose sequence and functions have been well conserved in evolution. All DNA damage response requires either ATM or ATR because they have the ability to bind to the chromosomes at the site of DNA damage, together with accessory proteins that are platforms on which DNA damage response components and DNA repair complexes can be assembled. An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage.[51] The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent kinase complexes.[52] The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes.[53] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes.[54] The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed by DNA helicase to separate the two DNA strands.[38] In the initiation step, RecA protein binds to ssDNA in an ATP hydrolysis driven reaction creating RecA–ssDNA filaments. RecA–ssDNA filaments activate LexA autoprotease activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division and an increase in levels of proteins responsible for damage processing. In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably, with different length and composition, but it is always highly conserved and one of the strongest short signals in the genome.[54] The high information content of SOS boxes permits differential binding of LexA to different promoters and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response. The error-prone translesion polymerases, for example, UmuCD'2 (also called DNA polymerase V), are induced later on as a last resort.[55] Once the DNA damage is repaired or bypassed using polymerases or through recombination, the amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene expression. Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage differently indicating an absence of a common global response. The probable explanation for this difference between yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage.[56] In general global response to DNA damage involves expression of multiple genes responsible for postreplication repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations. Yeast Rev1 and human polymerase η are members of [Y family translesion DNA polymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes.[38] Main article: DNA damage theory of aging DNA repair rate is an important determinant of cell pathology Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence.[13] For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice.[57] In similar manner, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan.[58] However, not every DNA repair deficiency creates exactly the predicted effects; mice deficient in the NER pathway exhibited shortened life span without correspondingly higher rates of mutation.[59] If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging,[13] increased sensitivity to carcinogens, and correspondingly increased cancer risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.[60] Most life span influencing genes affect the rate of DNA damage A number of individual genes have been identified as influencing variations in life span within a population of organisms. The effects of these genes is strongly dependent on the environment, in particular, on the organism's diet. Caloric restriction reproducibly results in extended lifespan in a variety of organisms, likely via nutrient sensing pathways and decreased metabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see[61] for some discussion); however, the behavior of many genes known to be involved in DNA repair is altered under conditions of caloric restriction. For example, increasing the gene dosage of the gene SIR-2, which regulates DNA packaging in the nematode worm Caenorhabditis elegans, can significantly extend lifespan.[62] The mammalian homolog of SIR-2 is known to induce downstream DNA repair factors involved in NHEJ, an activity that is especially promoted under conditions of caloric restriction.[63] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents,[64] although similar effects have not been observed in mitochondrial DNA.[65] The C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under conditions of caloric restriction.[66] This observation supports the pleiotropy theory of the biological origins of aging, which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a corresponding disadvantage late in life. Main article: DNA repair-deficiency disorder Defects in the NER mechanism are responsible for several genetic disorders, including: Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons. Other DNA repair disorders include: All of the above diseases are often called "segmental progerias" ("accelerated aging diseases") because their victims appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the symptoms of old age. Other diseases associated with reduced DNA repair function include Fanconi anemia, hereditary breast cancer and hereditary colon cancer. Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer.[67][68] There are at least 34 Inherited human DNA repair gene mutations that increase cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCA1 and BRCA2, two famous genes whose mutations confer a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing — most typically cancer cells — are preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as progenitor cells in the gut, skin, and hematopoietic system are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). …; in the context of therapies targeting DNA damage response genes, the latter approach has been termed ‘synthetic lethality’.[69] Perhaps the most well-known of these 'synthetic lethality' drugs is the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, which was approved by the Food and Drug Administration in 2015 for the treatment in women of BRCA-defective ovarian cancer. Tumor cells with partial loss of DNA damage response (specifically, homologous recombination repair) are dependent on another mechanism – single-strand break repair – which is a mechanism consisting, in part, of the PARP1 gene product.[70] Olaparib is combined with chemotherapeutics to inhibit single-strand break repair induced by DNA damage caused by the co-administered chemotherapy. Tumor cells relying on this residual DNA repair mechanism are unable to repair the damage and hence are not able to survive and proliferate, whereas normal cells can repair the damage with the functioning homologous recombination mechanism. Many other drugs for use against other residual DNA repair mechanisms commonly found in cancer are currently under investigation. However, synthetic lethality therapeutic approaches have been questioned due to emerging evidence of acquired resistance, achieved through rewiring of DNA damage response pathways and reversion of previously-inhibited defects.[71] It has become apparent over the past several years that the DNA damage response acts as a barrier to the malignant transformation of preneoplastic cells.[72] Previous studies have shown an elevated DNA damage response in cell-culture models with oncogene activation[73] and preneoplastic colon adenomas.[74] DNA damage response mechanisms trigger cell-cycle arrest, and attempt to repair DNA lesions or promote cell death/senescence if repair is not possible. Replication stress is observed in preneoplastic cells due to increased proliferation signals from oncogenic mutations. Replication stress is characterized by: increased replication initiation/origin firing; increased transcription and collisions of transcription-replication complexes; nucleotide deficiency; increase in reactive oxygen species (ROS).[75] Replication stress, along with the selection for inactivating mutations in DNA damage response genes in the evolution of the tumor,[76] leads to downregulation and/or loss of some DNA damage response mechanisms, and hence loss of DNA repair and/or senescence/programmed cell death. In experimental mouse models, loss of DNA damage response-mediated cell senescence was observed after using a short hairpin RNA (shRNA) to inhibit the double-strand break response kinase ataxia telangiectasia (ATM), leading to increased tumor size and invasiveness.[74] Humans born with inherited defects in DNA repair mechanisms (for example, Li-Fraumeni syndrome) have a higher cancer risk.[77] The prevalence of DNA damage response mutations differs across cancer types; for example, 30% of breast invasive carcinomas have mutations in genes involved in homologous recombination.[72] In cancer, downregulation is observed across all DNA damage response mechanisms (base excision repair (BER), nucleotide excision repair (NER), DNA mismatch repair (MMR), homologous recombination repair (HR), non-homologous end joining (NHEJ) and translesion DNA synthesis (TLS).[78] As well as mutations to DNA damage repair genes, mutations also arise in the genes responsible for arresting the cell cycle to allow sufficient time for DNA repair to occur, and some genes are involved in both DNA damage repair and cell cycle checkpoint control, for example ATM and checkpoint kinase 2 (CHEK2) – a tumor suppressor that is often absent or downregulated in non-small cell lung cancer.[79] Table: Genes involved in DNA damage response pathways and frequently mutated in cancer (HR = homologous recombination; NHEJ = non-homologous end joining; SSA = single-strand annealing; FA = fanconi anemia pathway; BER = base excision repair; NER = nucleotide excision repair; MMR = mismatch repair) Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations.[80] Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification,[81] changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1)[82] and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations). While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers.[83][84][85][86] Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair[87][88] or in homologous recombinational repair (HRR).[89] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.[90] Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing.[91][92] Deficient expression of DNA repair proteins due to an inherited mutation can cause increased risk of cancer. Individuals with an inherited impairment in any of 34 DNA repair genes (see article DNA repair-deficiency disorder) have an increased risk of cancer, with some defects causing up to a 100% lifetime chance of cancer (e.g. p53 mutations).[93] However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.[94] A chart of common DNA damaging agents, examples of lesions they cause in DNA, and pathways used to repair these lesions. Also shown are many of the genes in these pathways, an indication of which genes are epigenetically regulated to have reduced (or increased) expression in various cancers. It also shows genes in the error prone microhomology-mediated end joining pathway with increased expression in various cancers. Deficiencies in DNA repair enzymes are occasionally caused by a newly arising somatic mutation in a DNA repair gene, but are much more frequently caused by epigenetic alterations that reduce or silence expression of DNA repair genes. For example, when 113 colorectal cancers were examined in sequence, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration).[95] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.[96][97][98][99][100] Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1).[101] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.[102] In further examples (tabulated in Table 4 of this reference[103]), epigenetic defects were found at frequencies of between 13%–100% for the DNA repair genes BRCA1, WRN, FANCB, FANCF, MGMT, MLH1, MSH2, MSH4, ERCC1, XPF, NEIL1 and ATM. These epigenetic defects occurred in various cancers (e.g. breast, ovarian, colorectal and head and neck). Two or three deficiencies in the expression of ERCC1, XPF or PMS2 occur simultaneously in the majority of the 49 colon cancers evaluated by Facista et al.[104] The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes.[105] Of these, 83 are directly employed in repairing the 5 types of DNA damages illustrated in the chart. Some of the more well studied genes central to these repair processes are shown in the chart. The gene designations shown in red, gray or cyan indicate genes frequently epigenetically altered in various types of cancers. Wikipedia articles on each of the genes highlighted by red, gray or cyan describe the epigenetic alteration(s) and the cancer(s) in which these epimutations are found. Two review articles,[103][106] and two broad experimental survey articles[107][108] also document most of these epigenetic DNA repair deficiencies in cancers. Red-highlighted genes are frequently reduced or silenced by epigenetic mechanisms in various cancers. When these genes have low or absent expression, DNA damages can accumulate. Replication errors past these damages (see translesion synthesis) can lead to increased mutations and, ultimately, cancer. Epigenetic repression of DNA repair genes in accurate DNA repair pathways appear to be central to carcinogenesis. The two gray-highlighted genes RAD51 and BRCA2, are required for homologous recombinational repair. They are sometimes epigenetically over-expressed and sometimes under-expressed in certain cancers. As indicated in the Wikipedia articles on RAD51 and BRCA2, such cancers ordinarily have epigenetic deficiencies in other DNA repair genes. These repair deficiencies would likely cause increased unrepaired DNA damages. The over-expression of RAD51 and BRCA2 seen in these cancers may reflect selective pressures for compensatory RAD51 or BRCA2 over-expression and increased homologous recombinational repair to at least partially deal with such excess DNA damages. In those cases where RAD51 or BRCA2 are under-expressed, this would itself lead to increased unrepaired DNA damages. Replication errors past these damages (see translesion synthesis) could cause increased mutations and cancer, so that under-expression of RAD51 or BRCA2 would be carcinogenic in itself. Cyan-highlighted genes are in the microhomology-mediated end joining (MMEJ) pathway and are up-regulated in cancer. MMEJ is an additional error-prone inaccurate repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5–25 complementary base pairs between both paired strands is sufficient to align the strands, but mismatched ends (flaps) are usually present. MMEJ removes the extra nucleotides (flaps) where strands are joined, and then ligates the strands to create an intact DNA double helix. MMEJ almost always involves at least a small deletion, so that it is a mutagenic pathway.[109] FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast,[110] prostate,[111] stomach,[112][113] neuroblastomas,[114] pancreas,[115] and lung.[116] PARP1 is also over-expressed when its promoter region ETS site is epigenetically hypomethylated, and this contributes to progression to endometrial cancer,[117] BRCA-mutated ovarian cancer,[118] and BRCA-mutated serous ovarian cancer.[119] Other genes in the MMEJ pathway are also over-expressed in a number of cancers (see MMEJ for summary), and are also shown in cyan. Differential activity of DNA repair pathways across various regions of the human genome causes mutations to be very unevenly distributed within tumor genomes.[120][121] In particular, the gene-rich, early-replicating regions of the human genome exhibit lower mutation frequencies than the gene-poor, late-replicating heterochromatin. One mechanism underlying this involves the histone modification H3K36me3, which can recruit mismatch repair proteins,[122] thereby lowering mutation rates in H3K36me3-marked regions.[123] Another important mechanism concerns nucleotide excision repair, which can be recruited by the transcription machinery, lowering somatic mutation rates in active genes[121] and other open chromatin regions.[124] The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophages (viruses which infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.[125] The ability of a large number of protein structural motifs to catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see.[126] The fossil record indicates that single-cell life began to proliferate on the planet at some point during the Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic form have been inherited by all extant life forms from their common ancestor. The emergence of Earth's oxygen-rich atmosphere (known as the "oxygen catastrophe") due to photosynthetic organisms, as well as the presence of potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced by oxidative stress. On some occasions, DNA damage is not repaired, or is repaired by an error-prone mechanism that results in a change from the original sequence. When this occurs, mutations may propagate into the genomes of the cell's progeny. Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential to be passed on to the organism's offspring. The rate of evolution in a particular species (or, in a particular gene) is a function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an influence over the process of evolutionary change.[127] Since the normal adaptation of populations of organisms to changing circumstances (for instance the adaptation of the beaks of a population of finches to the changing presence of hard seeds or insects) proceeds by gene regulation and the recombination and selection of gene variations – alleles – and not by passing on irreparable DNA damages to the offspring,[128] DNA damage protection and repair does not influence the rate of adaptation by gene regulation and by recombination and selection of alleles. On the other hand, DNA damage repair and protection does influence the rate of accumulation of irreparable, advantageous, code expanding, inheritable mutations, and slows down the evolutionary mechanism for expansion of the genome of organisms with new functionalities. The tension between evolvability and mutation repair and protection needs further investigation. A technology named clustered regularly interspaced short palindromic repeat shortened to CRISPR-Cas9 was discovered in 2012. The new technology allows anyone with molecular biology training to alter the genes of any species with precision.[129] Listen to this article (info/dl) This audio file was created from a revision of the article "DNA repair" dated 2005-06-17, and does not reflect subsequent edits to the article. (Audio help) More spoken articles

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"Toolshed" redirects here. For the Tool demo album, see 72826. "Bike shed" redirects here. For bike-shedding, see law of triviality. A rural shed Modern secure bike sheds Garden shed with gambrel roof A shed is typically a simple, single-storey roofed structure in a back garden or on an allotment that is used for storage, hobbies, or as a workshop. Sheds vary considerably in the complexity of their construction and their size, from small open-sided tin-roofed structures to large wood-framed sheds with shingled roofs, windows, and electrical outlets. Sheds used on farms or in industry can be large structures. The main types of shed construction are metal sheathing over a metal frame, plastic sheathing and frame, all-wood construction (the roof may be asphalt shingled or sheathed in tin), and vinyl-sided sheds built over a wooden frame. A culture of shed enthusiasts exists in several countries for people who enjoy building sheds and spending time in them for relaxation. In Australia and New Zealand there are magazines called The Shed, an association for shed hobbyists (the Australian Men's Shed Association), and a book entitled Men and Sheds. Depending on the region and type of use, a shed may also be called an "outhouse", "outbuilding" or "shack". The simplest and least-expensive sheds are available in kit form. These kits are designed for regular people to be able to assemble themselves using commonly available tools (e.g., screwdriver). Both shed kits and DIY (do-it-yourself) plans are available for wooden and plastic sheds. Sheds are used to store home and garden tools and equipment such as lawn tractors, and gardening supplies. In addition, sheds can be used to store items that are not suitable for indoor storage, such as petrol (gasoline), pesticides, or herbicides. For homes with small gardens or modest storage needs, there are several types of very small sheds. The sheds not only use less ground area but also have a low profile less likely to obstruct the view or clash with the landscaping. A metal garden shed made with sheets of galvanized steel over a steel frame These small sheds include corner sheds, which fit into a corner (3 ft tall × 3 wide × 2 deep, or 0.91 m × 0.91 m × 0.61 m), vertical sheds (5 ft × 3 ft × 4 ft deep, or 1.52 m × 0.91 m × 1.22 m), horizontal sheds (3 ft × 5 ft × 4 ft or 0.91 m × 1.52 m × 1.22 m), and tool sheds. When a shed is used for tool storage, shelves and hooks are often used to maximize the storage space. Gambrel-style roofed sheds (sometimes called baby barns), which resemble a Dutch-style barn, have a high sloping roofline which increases storage space in the "loft" area. Some Gambrel-styles have no loft and offer the advantage of reduced overall height. Another style of small shed is the saltbox-style shed. Many sheds have either a pent or apex roof shape. A pent shed features a single roof section which is angled downwards to let rainwater run off, with more headroom at the front than the back. This is a simple, practical design that will fit particularly well next to a wall or fence. It is also usually lower than the typical apex shed, so could be a better choice if there are any height restrictions. A pent shed may be free-standing or attached to a wall (when it is known, unsurprisingly, as a wall shed). An apex shed has a pointed roof in an inverted V shape similar to the roof line of many houses. Two roof sections meet at a ridge in the middle, providing more headroom in the centre than at the sides. This type is generally regarded as a more attractive and traditional design, and may be preferable if the shed is going to be visible from the house. [1] A twist on the standard apex shape is the reverse apex shed. In this design, the door is set in a side wall instead of the front. The main advantage of the reverse apex design is that the door opens into the widest part of the shed instead of the narrowest, so it's easier to reach into all areas to retrieve or store equipment. [2] A tall shed with windows and a shingled roof Larger, more-expensive sheds are typically constructed of wood and include features typically found in house construction, such as windows, a shingled roof, and electrical outlets. Larger sheds provide more space for engaging in hobbies such as gardening, small engine repair, or tinkering. Some sheds have small porches or include furniture, which allows them to be used for relaxation purposes. In some cases, teleworkers and homeworkers in general who live in mild climates use small to medium-sized wooden garden sheds as outdoor offices. There is a growing industry in providing "off the peg" garden offices to cater for this demand, particularly in the UK but also in the US. Shed owners can customize wooden sheds to match the features (e.g., siding, trim, etc.) of the main house. A number of decorative options can be added to sheds, such as dormers, shutters, flowerboxes, finials, and weathervanes. As well, practical options can be added such as benches, ramps, ventilation systems (e.g., in cases where a swimming pool heater is installed in a shed), and electric lighting. Sheds designed for gardening, called "potting sheds", often feature windows or skylights for illumination, ventilation grilles, and a potter's bench for mixing soil and re-potting plants. "Bicycle shed" redirects here. For "the bicycle shed effect", see Law of triviality. A bike shed The main types of shed construction are metal sheathing over a metal frame, plastic sheathing and frame, all-wood construction (wood frame, wood siding and wood roof), and vinyl-sided sheds built over a wooden frame. Each type has various advantages and disadvantages that a homeowner has to consider.[4] For example, while metal sheds are fire and termite-resistant, they can rust over time, or be severely damaged by high winds or heavy snow loads. Wood sheds are easier to modify or customize than plastic or metal, because carpentry tools and basic carpentry skills are more readily available. Vinyl-sided, wood-framed sheds blend the strength of a wood frame with the maintenance-free aspect of vinyl siding (it does not need to be painted or varnished). The International Building Code (IBC) defines a shed as a building or structure of an accessory character; it classifies them under utility and miscellaneous group U (Chapter 3 Section 312). A corrugated iron shed Metal sheds made from thin sheet metal sheathing (galvanized steel, aluminium, or corrugated iron) attached to a metal frame. Metal sheds are a good choice when long-term strength and resistance to fire, rot, or termites is desired. However, metal sheds may rust over time, particularly if they are constructed from steel that is not galvanized. Be aware that concrete is highly corrosive so care needs to be taken when assembling your shed to avoid contact with the outside panels.[5] As well, some types of metal sheds that have thin walls are easily dented, which may makes some types of thin metal sheds a poor choice for vandal-prone areas or for high-traffic activities such as small businesses. In cold climates, metal sheds with thin walls need to have snow and ice cleared from the roof, because the thin metal may be damaged by a heavy accumulation. Since thin metal sheds weigh much less than wood or PVC plastic sheds, thin metal sheds are more at risk of being damaged by heavy winds. To prevent wind damage, thin metal sheds should be attached to a concrete foundation with screws.[6] In countries where the climate is generally mild, such as Australia, very large metal sheds are used for many types of industry. Corrugated metal sheds may be better able to withstand wind and snow loads, as the corrugated shape makes the metal stronger than flat tin. Lifetime brand blow-molded plastic sheds Plastic shed kits utilizing heavy molded plastics such as PVC and polyethylene may be less expensive than sheet-metal sheds. PVC resins and high-impact, UV light-resistant polyethylene make plastic outdoor sheds stronger, lighter, more durable, and more resistant to denting and chipping than wood, and tend to be more stable. Plastic shed kits sided with vinyl are typically among the least-expensive types of shed construction. Higher-quality sheds use UV-resistant plastic and powder-coated metal frames. Many plastic sheds are modular to allow for easy extensions, peg-boards, shelving, attic-storage, windows, skylights, and other accessories to be added later, if these additions are purchased from the manufacturer. Plastic sheds are not susceptible to termite or wood-boring insect damage, and they require little maintenance. Being rot-proof they do not need to have preservative applied. This makes them preferable in climates where the weather can be changeable, such as the United Kingdom.[7] Unlike wooden or metal sheds, which often require a permit to build, in many areas, plastic sheds do not. However, this is something property owners will need to verify. A call to your council/town's planning or building code office can provide information on permits.[8] Domestic wooden sheds. Example of wood storage shed from US cedar shed builder. Wooden sheds have a natural look that can blend in well with garden environments. Despite the strength of wood, over time, untreated and neglected wood can rot, split, warp or become susceptible to mold and mildew, so wood sheds should be treated for protection with stain and varnish. Wood sheds need regular maintenance. This includes keeping plant matter and debris from piling up beside the walls and on the roof, and occasional rot-proofing with preservative. Sheds are sometimes also re-stained or varnished at times for aesthetic and wood protection reasons. Fire and, in some regions, termite attack are also potential problems. Stains and preservatives can be applied to wood sheds to prevent damage to the wood caused by exposure to rain, damp ground, UV light, harsh climatic conditions, fungal attack and wood-boring insects. If a coloured preservative oil or stain is used, a wooden shed can either be made to stand out as a feature within a garden, or to blend in with its surroundings. Red cedar coloured stain is popular. Some types of wood, such as cedar, are more naturally resistant to water damage. When looking for a wooden shed, it is important to understand the difference between the two types of preservative used in their manufacture. The timber will have been treated in one of two ways: dip treatment and pressure treatment. Dip-treated sheds are made from components that are lowered into a tank of preservative before the panels are assembled. This is a quick and simple process which keeps costs down and encourages manufacturers to produce a wide variety, making dip-treated sheds the most popular and affordable type on the market. They are easily recognisable by their golden brown colour, which is due to a dye added to the preservative. Most manufacturers offer a 10-year anti-rot guarantee on dip-treated sheds, but they have to be re-coated every year or two. [2] Pressure-treated sheds are made from timber planks which have had the moisture sucked out of them under vacuum conditions in a special cylinder. A powerful preservative is then forced into the wood at high pressure until it is absorbed deep into the grain, becoming an integral part of the timber. This provides excellent protection against the weather - so much so that manufacturers generally give a 15-year anti-rot guarantee. These sheds are usually distinguished by a pale green tinge which will fade eventually to a silvery grey. Although pressure-treated sheds tend to be more expensive than dip-treated ones, their big advantage is that they won't need any further preservative treatment during the guarantee period, saving owners time and money. [3] One advantage of using wood sheds over metal versions is that it is easier to modify them by adding windows, doors, shelving, or exterior trim (etc.) because wood can be cut and drilled using commonly available tools, whereas a plastic or metal shed requires specialized tools. Some homeowners may prefer wood sheds because wood is a renewable resource. An Amish-style vinyl-sided shed Vinyl-sided sheds are typically built with standard wood framing construction and oriented strand board (OSB) on the walls covered with standard vinyl siding. The vinyl siding protects the OSB wood and the frame from moisture from rain and snow. Vinyl-sided sheds never need to be painted, and are maintenance-free. They are stronger than plastic or metal sheds, and are usually built to conform with the local building codes. They offer good value for money because they hold up in all weather, including winters with heavy snowfall, as they use a strong wooden frame and the OSB panels have stronger structural support than thin metal or PVC siding or roofs. Metal, plastic and resin sheds are cheaper, but they cannot handle the weight of snow in winter (roofs may cave in). Vinyl sheds also offer more colour options. In the early and middle years of the 20th century, many garden sheds and domestic garages were made of asbestos-cement sheets supported on a very light angle-iron frame. Concerns about safety led to the practice being discontinued, but they were cheap and long-lasting, and many can still be seen in British gardens. Advice on continued use or disposal is available.[9] Since 2013 garden sheds have been available in the UK made from TPR - a sustainable alternative to concrete.[10] They are typically coated in a marine gelcoat and are far stronger and more durable than traditional sheds. A shed made from TPR became the first Secured by Design-approved shed in 2014[11] A shed near Sydney, Australia In Australia and New Zealand the term shed can be used to refer to any building that is not a residence and which may be open at the ends or sides, or both. Australia's passion for sheds is documented in Mark Thomson's Blokes and Sheds (1998).[12] Jim Hopkins' similarly titled Blokes & Sheds (1998), with photographer Julie Riley Hopkins, profiles amateur inventors from across New Zealand.[13] Hopkins and Riley followed up that book with Inventions from the Shed (1999)[14] and a 5-part film documentary series with the same name.[15] Gordon Thorburn also examined the shed proclivity in his book Men and Sheds (2002),[16] as did Gareth Jones in Shed Men (2004).[17] Recently, "Men's Sheds" have become common in Australia.[18] In New Zealand, the bi-monthly magazine The Shed appeals to the culture of "blokes" who do woodwork or metalwork DIY projects in their sheds. The Australian Men's Shed Association is one organisation that has been set up involving sheds. A much-loved and frequently restored British shed in Lincolnshire Another magazine called The Shed, a bimonthly PDF magazine produced in the UK, but with a global audience, targets people who work (usually in creative industries) in garden offices, sheds and other shed-like atmospheres.[citation needed] In the UK, people have long enjoyed working in their potting sheds; the slang term "sheddie", to refer to a person enamoured of shed-building, testifies to the place of sheds in UK popular culture. A Usenet Newsgroup "uk.rec.sheds" has long championed this subculture: their lengthy FAQ[19] is a masterly summary of the idea. Shedworking: A lifestyle guide for shedworkers is published at Blogger. Author Gordon Thorburn examined the shed proclivity in his book Men and Sheds, which argues that a "place of retreat" is a "male necessity" which provides men with solace, especially during their retirement.[20] In contrast, in the novel Cold Comfort Farm by Stella Gibbons, Aunt Edna Doom saw "something nasty in the woodshed" and retreated to her bed for half a century. To woodshed, or 'shed, in jazz jargon, is "to shut oneself up, away from the world, and practice long and hard, as in 'going to the woodshed'."[21] The word is recorded in English since 1481, as shadde, possibly a variant of shade. The word shade comes from the Old English word "sceadu", which means "shade, shadow, darkness". The term's P.Gmc. cognate, "skadwo" also means "shady place, protection from glare or heat".[22] The Old English word is spelled in different ways, such as "shadde", "shad" or "shedde", all of which come from an "Old Teutonic/Anglo-Saxon root word for separation or division". The first attested usage of the word, in 1481, was in the sentence, "A yearde in whiche was a shadde where in were six grete dogges". The Anglo Saxon word "shud", which means "cover" may also have been part of the development of the word. In 1440, a "shud" was defined as a "... schudde, hovel, swyne kote or howse of sympyl hyllynge [covering] to kepe yn beestys".[citation needed] A waterside shed in Sweden

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