Gene therapy

From Wikipedia, the free encyclopedia

Gene therapy using an adenovirus vector. A new gene is inserted into a cell using an adenovirus. If the treatment is successful, the new gene will make a functional protein to treat a disease.

Gene therapy is the therapeutic delivery of nucleic acid polymers into a patient's cells as a drug to treat disease. The polymers are either expressed as proteins, interfere with protein expression, or possibly correct genetic mutations.

The most common form uses DNA that encodes a functional, therapeutic gene to replace a mutated gene. The polymer molecule is packaged within a "vector", which carries the molecule inside cells.

Gene therapy was conceptualized in 1972, by authors who urged caution before commencing human gene therapy studies. The first gene therapy experiment approved by the US Food and Drug Administration (FDA) occurred in 1990, when Ashanti DeSilva was treated for ADA-SCID.^[1] By January 2014, some 2,000 clinical trials had been conducted or approved.^[2]

Early clinical failures led to dismissals of gene therapy. Clinical successes since 2006 regained researchers' attention, although as of 2014, it was still largely an experimental technique.^[3] These include treatment of retinal disease Leber's congenital amaurosis,^[4][5][6][7] X-linked SCID,^[8] ADA-SCID,^[9][10] adrenoleukodystrophy,^[11] chronic lymphocytic leukemia (CLL),^[12] acute lymphocytic leukemia (ALL),^[13] multiple myeloma,^[14] haemophilia^[10] and Parkinson's disease.^[15] Between 2013 and April 2014, US companies invested over $600 million in the field.^[16]

The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of certain cancers.^[17] In 2012 Glybera, a treatment for a rare inherited disorder, became the treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission.^[3][18]

Approaches

Following early advances in genetic engineering of bacteria, cells and small animals, scientists started considering how to apply it to to medicine. Two main approaches were considered – replacing or disrupting defective genes.^[19] Scientists focused on diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy, thalassemia and sickle cell anemia. Glybera treats one such disease, caused by a defect in lipoprotein lipase.^[18]

DNA must be administered, reach the damaged cells, enter the cell and express/disrupt a protein.^[20] Multiple delivery techniques have been explored. The initial approach incorporated DNA into an engineered virus to deliver the DNA into a chromosome.^[21][22] Naked DNA approaches have also been explored, especially in the context of vaccine development.^[23]

Generally, efforts focused on administering a gene that causes a needed protein to be expressed. More recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as zinc finger nucleases and CRISPR. The vector incorporates genes into chromosomes. The expressed nucleases then "edit" the chromosome. As of 2014 these approaches involve removing cells from patients, editing a chromosome and returning the transformed cells to patients.^[24]

Other technologies employ antisense, small interfering RNA and other DNA. To the extent that these technologies do not alter DNA, but instead directly interact with molecules such as RNA, they are not considered "gene therapy" per se.^{[citation needed]}

Cell types

Gene therapy may be classified into two types:

Somatic cell

In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any of any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Any such modifications affect the individual patient only, and are not inherited by offspring. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease.
Over 600 clinical trials utilizing SCGT are underway in the US. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy. The complete correction of a genetic disorder or the replacement of multiple genes is not yet possible. Only a few of the trials are in the advanced stages.^[25]

Germline

In germline gene therapy (GGT), germ cells (sperm or eggs) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations. Australia, Canada, Germany, Israel, Switzerland and the Netherlands^[26] prohibit GGT for application in human beings, for technical and ethical reasons, including insufficient knowledge about possible risks to future generations^[26] and higher risks versus SCGT.^[27] The US has no federal controls specifically addressing human genetic modification (beyond FDA regulations for therapies in general).^{[26][28][29][30]}

Vectors

The delivery of DNA into cells can be accomplished by multiple methods. The two major classes recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or DNA complexes (non-viral methods).

Viruses

Viruses introduce their genetic material into the host cell as part of their replication cycle. Viral vectors exploit this behavior by removing the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA.A number of viruses have been used for human gene therapy, including retrovirus, adenovirus, lentivirus, herpes simplex, vaccinia and adeno-associated virus.^[2]

Non-viral

Non-viral methods present certain advantages over viral methods, such as large scale production and low host immunogenicity. However, initially non-viral methods produced lower levels of transfection and gene expression. Later technology remedied this deficiency.

Methods for non-viral gene therapy include the injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles, zinc fingers and CRISPR.

Hurdles

Some of the unsolved problems include:

• Short-lived nature – Before gene therapy can become a permanent cure for a condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent it from achieving long-term benefits. Patients require multiple treatments.
• Immune response – Any time a foreign object is introduced into human tissues, the immune system is stimulated to attack the invader. Stimulating the immune system in a way that reduces gene therapy effectiveness is possibile. The immune system's enhanced response to viruses that it has seen before reduces the effectiveness to repeated treatments.
• Problems with viral vectors – Viral vectors carry the risks of toxicity, inflammatory responses, and gene control and targeting issues. The virus also may recover its ability to cause disease.
• Multigene disorders – Some commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are affected by variations in multiple genes, which complicate gene therapy.
• Some therapies may breach the Weismann barrier (between soma and germ-line) protecting the testes, potentially modifying the germline, falling afoul of regulations in countries that prohibit the latter practice.^[31]
• Insertional mutagenesis – If the DNA is integrated in a sensitive spot in the genome, for example in a tumor suppressor gene, the therapy could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.^[32][33] One possible solution is to add a functional tumor suppressor gene to the DNA to be integrated. This may be problematic since the longer the DNA is, the harder it is to integrate into cell genomes. CRISPR technology allows researchers to make much more precise genome changes at exact locations.^[34]
• Cost – Alipogene tiparvovec or Glybera, for example, at a cost of $1.6 million per patient, was reported in 2013 to be the world's most expensive drug.^[35][36]

Deaths

Three patients' deaths have been reported in gene therapy trials, putting the field under close scrutiny. The first was that of Jesse Gelsinger in 1999.^[37] One X-SCID patient died of leukemia in 2003.^[1] In 2007, a rheumatoid arthritis patient died from an infection; the subsequent investigation concluded that the death was not related to gene therapy.^[38]

History

1970s and earlier

In 1972 Friedmann and Roblin authored a paper in Science titled "Gene therapy for human genetic disease?"^[39] Rogers (1970) was cited for proposing that exogenous good DNA be used to replace the defective DNA in those who suffer from genetic defects.^[40]

1980s

In 1984 a retrovirus vector system was designed that could efficiently insert foreign genes into mammalian chromosomes.^[41]

1990s

The first approved gene therapy in the US took place on 14 September 1990, at the National Institutes of Health (NIH), under the direction of William French Anderson.^[42] Four-year-old Ashanti DeSilva received treatment for a genetic defect that left her with ADA-SCID, a severe immune system deficiency. The effects were temporary, but successful.^[43]

Cancer therapy of glioblastoma multiforme, the most common human brain tumor, whose outcome is always fatal, was introduced in 1992/93y.^[44] The strategy used a vector expressing antisense IGF-I RNA, showed promising results in clinical trials (approved by NIH n˚ 1602, and FDA in 1994). This strategy proved to be effective due to the anti-tumor mechanism of IGF-I antisense, which is related to strong immune and apoptotic phenomena. The median survival reached 21 months, and in some cases, three to four years.^[45]

In 1992 Claudio Bordignon, working at the Vita-Salute San Raffaele University, performed the first gene therapy procedure using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases.^[46] In 2002 this work led to the publication of the first successful gene therapy treatment for adenosine deaminase-deficiency (SCID). The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) from 2000 and 2002, was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the US, the United Kingdom, France, Italy and Germany.^[47]

In 1993 Andrew Gobea was born with SCID following prenatal genetic screening. Blood was removed from his mother's placenta and umbilical cord immediately after birth, to acquire stem cells. The allele that codes for adenosine deaminase (ADA) was obtained and inserted into a retrovirus. Retroviruses and stem cells were mixed, after which the viruses inserted the gene into the stem cell chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood. Injections of the ADA enzyme were also given weekly. For four years T cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed.^{[citation needed]}

Jesse Gelsinger 's death in 1999 impeded gene therapy research in the US.^[48][49] As a result, the FDA suspended several clinical trials pending the reevaluation of ethical and procedural practices.^[50]

2000s

2002

Sickle-cell disease can be treated in mice.^[51] The mice – which have essentially the same defect that causes human cases – used a viral vector to induce production of fetal hemoglobin (HbF), which normally ceases to be produced shortly after birth. In humans, the use of hydroxyurea to stimulate the production of HbF temporarily alleviates sickle cell symptoms. The researchers demonstrated this treatment to be a more permanent means to increase therapeutic HbF production.^[52]

A new gene therapy approach repaired errors in messenger RNA derived from defective genes. This technique has the potential to treat thalassaemia, cystic fibrosis and some cancers.^[53]

Researchers created liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane.^[54]

2003

In 2003 a research team inserted genes into the brain for the first time. They used liposomes coated in a polymer called polyethylene glycol, which, unlike viral vectors, are small enough to cross the blood–brain barrier.^[55]

Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced.^[56]

Gendicine is a cancer gene therapy that delivers the tumor suppressor gene p53 using an engineered adenovirus. In 2003, it was approved in China for the treatment of head and neck squamous cell carcinoma.^[17]

2006

In March researchers announced the successful use of gene therapy to treat two adult patients for X-linked chronic granulomatous disease, a disease which affects myeloid cells and damages the immune system. The study is the first to show that gene therapy can treat the myeloid system.^[57]

In May a team reported a way to prevent the immune system from rejecting a newly delivered gene.^[58] Similar to organ transplantation, gene therapy has been plagued by this problem. The immune system normally recognizes the new gene as foreign and rejects the cells carrying it. The research utilized a newly uncovered network of genes regulated by molecules known as microRNAs. This natural function selectively obscured their therapeutic gene in immune system cells and protected it from discovery. Mice infected with the gene containing an immune-cell microRNA target sequence did not reject the gene.

In August scientists successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells.^[59]

In November researchers reported on the use of VRX496, a gene-based immunotherapy for the treatment of HIV that uses a lentiviral vector to deliver an antisense gene against the HIV envelope. In a phase I clinical trial, five subjects with chronic HIV infection who had failed to respond to at least two antiretroviral regimens were treated. A single intravenous infusion of autologous CD4 T cells genetically modified with VRX496 was well tolerated. All patients had stable or decreased viral load; four of the five patients had stable or increased CD4 T cell counts. All five patients had stable or increased immune response to HIV antigens and other pathogens. This was the first evaluation of a lentiviral vector administered in a US human clinical trial.^[60][61]

2007

In May researchers announced the first gene therapy trial for inherited retinal disease. The first operation was carried out on a 23-year-old British male, Robert Johnson, in early 2007.^[62]

2008

Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of a small clinical trial in children were published in April.^[63] Delivery of recombinant adeno-associated virus (AAV) carrying RPE65 yielded positive results. In May two more groups reported positive results in independent clinical trials using gene therapy to treat the condition. In all three clinical trials, patients recovered functional vision without apparent side-effects.^[4][5][6][7]

2009

In September researchers were able to give trichromatic vision to squirrel monkeys.^[64] In November 2009, researchers halted a fatal genetic disorder called adrenoleukodystrophy in two children using a lentivirus vector to deliver a functioning version of ABCD1, the gene that is mutated in the disorder.^[65]

2010s

2010

An April paper reported that gene therapy addressed achromatopsia (color blindness) in dogs by targeting cone photoreceptors. Cone function and day vision were restored for at least 33 months in two young specimens. The therapy was less efficient for older dogs.^[66]

In September it was announced that an 18 year old male patient in France with beta-thalassemia major had been successfully treated.^[67] Beta-thalassemia major is an inherited blood disease in which beta haemoglobin is missing and patients are dependent on regular lifelong blood transfusions.^[68] The technique used a lentiviral vector to transduce the human ß-globin gene into purified blood and marrow cells obtained from the patient in June 2007.^[69]
The patient's haemoglobin levels were stable at 9 to 10 g/dL. About a third of the hemoglobin contained the form introduced by the viral vector and blood transfusions were not needed.^[69][70] Further clinical trials were planned.^[71] Bone marrow transplants are the only cure for thalassemia, but 75% of patients do not find a matching donor.^[70]

2011

In 2007 and 2008, a man was cured of HIV by repeated Hematopoietic stem cell transplantation (see also Allogeneic stem cell transplantation, Allogeneic bone marrow transplantation, Allotransplantation) with double-delta-32 mutation which disables the CCR5 receptor. This cure was accepted by the medical community in 2011.^[72] It required complete ablation of existing bone marrow, which is very debilitating.

In August two of three subjects of a pilot study were confirmed to have been cured from chronic lymphocytic leukemia (CLL). The therapy used genetically modified T cells to attack cells that expressed the CD19 protein to fight the disease.^[12] In 2013, the researchers announced that 26 of 59 patients had achieved complete remission and the original patient had remained tumor-free.^[73]

Human HGF plasmid DNA therapy of cardiomyocytes is being examined as a potential treatment for coronary artery disease as well as treatment for the damage that occurs to the heart after myocardial infarction.^[74][75]

2012

The FDA approved Phase 1 clinical trials on thalassemia major patients in the US for 10 participants in July.^[76] The study was expected to continue until 2015.^[77]

In July 2012, the European Medicines Agency recommended approval of a gene therapy treatment for the first time in either Europe or the United States. The treatment used Alipogene tiparvovec (Glybera) to compensate for lipoprotein lipase deficiency, which can cause severe pancreatitis.^[78] The recommendation was endorsed by the European Commission in November 2012^{[3][18][79][80]} and commercial rollout began in late 2014.^[81]

In December 2012, it was reported that 10 of 13 patients with multiple myeloma were in remission "or very close to it" three months after being injected with a treatment involving genetically engineered T cells to target proteins NY-ESO-1 and LAGE-1, which exist only on cancerous myeloma cells.^[14]

2013

In March researchers reported that three of five subjects who had acute lymphocytic leukemia (ALL) had been in remission for five months to two years after being treated with genetically modified T cells which attacked cells with CD19 genes on their surface, i.e. all B-cells, cancerous or not. The researchers believed that the patients' immune systems would make normal T-cells and B-cells after a couple of months. They were also given bone marrow. One patient relapsed and died and one died of a blood clot unrelated to the disease.^[13]

Following encouraging Phase 1 trials, in April, researchers announced they were starting Phase 2 clinical trials (called CUPID2 and SERCA-LVAD) on 250 patients^[82] at several hospitals to combat heart disease. The therapy was designed to increase the levels of SERCA2a protein in heart muscles, improving muscle function.^[83] The FDA granted this a Breakthrough Therapy Designation to accelerate the trial and approval process.^[84]

In July researchers reported promising results for six children with two severe hereditary diseases had been treated with a partially deactivated lentivirus to replace a faulty gene and after 7–32 months. Three of theb children had metachromatic leukodystrophy, which causes children to lose cognitive and motor skills.^[85] The other children had Wiskott-Aldrich syndrome, which leaves them to open to infection, autoimmune diseases and cancer.^[86]

In October researchers reported that two children born with adenosine deaminase severe combined immunodeficiency disease (ADA-SCID) had been treated with genetically engineered stem cells 18 months previously and that their immune systems were showing signs of full recovery. Another three children were making progress.^[10] In 2014 a further 18 children with ADA-SCID were cured by gene therapy.^[87] ADA-SCID children have no functioning immune system and are sometimes known as "bubble children."^[10]

Also in October researchers reported that they had treated six haemophilia sufferers in early 2011 using an adeno-associated virus. Over two years later all six were producing clotting factor.^[10][88]

Data from three trials on Topical cystic fibrosis transmembrane conductance regulator gene therapy were reported to not support its clinical use as a mist inhaled into the lungs to treat cystic fibrosis patients with lung infections.^[89]

2014

In January researchers reported that six choroideremia patients had been treated with adeno-associated virus with a copy of REP1. Over a six-month to two-year period all had improved their sight. Choroideremia is an inherited genetic eye disease no approved treatment, leading to loss of signt.^[90][91]

In March researchers reported that 12 HIV patients had been treated since 2009 in a trial with a genetically engineered virus with a rare mutation (CCR5 deficiency) known to protect against HIV with promising results.^[92][93]

Clinical trials of gene therapy for sickle cell disease were started in 2014^[94][95] although one review failed to find any such trials.^[96]

2015

In February LentiGlobin BB305, a gene therapy treatment undergoing clinical trials for treatment of beta thalassemia gained FDA "breakthrough" status after several patients were able to forgo the frequent blood transfusions usually required to treat the disease.^[97]

In March researchers delivered a recombinant gene encoding a broadly neutralizing antibody into monkeys infected with simian HIV; the monkey's cells produced the antibody, which cleared them of HIV. The technique is named immunoprophylaxis by gene transfer (IGT). Animal tests for antibodies to ebola, malaria, influenza and hepatitis are underway.^[98][99]

In March scientists, including an inventor of CRISPR, urged a worldwide moratorium on germline gene therapy, writing “scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans” until the full implications “are discussed among scientific and governmental organizations”.^{[100][101][102][103]}

Also in 2015 Glybera was approved for the German market.^[104]

Speculative uses

Speculated uses for gene therapy include:

Gene doping

Athletes might adopt gene therapy technologies to improve their performance.^[105] Gene doping is not known to occur, but multiple gene therapies may have such effects. Kayser et al. argue that gene doping could level the playing field if all athletes receive equal access. Critics claim that any therapeutic intervention for non-therapeutic/enhancement purposes compromises the ethical foundations of medicine and sports.^[106]

Human genetic engineering

Genetic engineering could be used to change physical appearance, metabolism, and even improve physical capabilities and mental faculties such as memory and intelligence. Ethical claims about germline engineering include beliefs that every fetus has a right to remain genetically unmodified, that parents hold such rights, and that every child has the right to be born free of preventable diseases.^{[107][108][109]} For adults, genetic engineering could be seen as another enhancement technique to add to diet, exercise, education, cosmetics and plastic surgery.^[110][111]
Another theorist claims that moral concerns limit but do not prohibit germline engineering.^[112]

Possible regulatory schemes include a complete ban, provision to everyone, or professional self-regulation. The American Medical Association’s Council on Ethical and Judicial Affairs stated that "genetic interventions to enhance traits should be considered permissible only in severely restricted situations: (1) clear and meaningful benefits to the fetus or child; (2) no trade-off with other characteristics or traits; and (3) equal access to the genetic technology, irrespective of income or other socioeconomic characteristics."^[113]

Regulations

Regulations covering genetic modification are part of general guidelines about human-involved biomedical research.

The Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects) was amended by the World Medical Association's General Assembly in 2008. This document provides principles physicians and researchers must consider when involving humans as research subjects. The Statement on Gene Therapy Research initiated by the Human Genome Organization (HUGO) in 2001 provides a legal baseline for all countries. HUGO’s document emphasizes human freedom and adherence to human rights, and offers recommendations for somatic gene therapy, including the importance of recognizing public concerns about such research.^[114]

United States

No federal legislation lays out protocols or restrictions about human genetic engineering. This subject is governed by overlapping regulations from local and federal agencies, including the Department of Health and Human Services, the FDA and NIH's Recombinant DNA Advisory Committee. Researchers seeking federal funds for an investigational new drug application, (commonly the case for somatic human genetic engineering), must obey international and federal guidelines for the protection of human subjects.^[115]

NIH serves as the main gene therapy regulator for federally funded research. Privately funded research is advised to follow these regulations. NIH provides funding for research that develops or enhances genetic engineering techniques and to evaluate the ethics and quality in current research. The NIH maintains a mandatory registry of human genetic engineering research protocols that includes all federally funded projects.

An NIH advisory committee published a set of guidelines on gene manipulation.^[116] The guidelines discuss lab safety as well as human test subjects and various experimental types that involve genetic changes. Several sections specifically pertain to human genetic engineering, including Section III-C-1. This section describes required review processes and other aspects when seeking approval to begin clinical research involving genetic transfer into a human patient.^[117]

The FDA regulates the quality and safety of gene therapy products and supervises how these products are used clinically. Therapeutic alteration of the human genome falls under the same regulatory requirements as any other medical treatment. Research involving human subjects, such as clinical trials, must be reviewed and approved by the FDA and an Institutional Review Board.^[118][119]

DNA repair (anti-aging / anti-cancer)

From Wikipedia, the free encyclopedia

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 UV light and 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]

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:

1. an irreversible state of dormancy, known as senescence
2. cell suicide, also known as apoptosis or programmed cell death
3. 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]

DNA damage

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.

Sources of damage

DNA damage can be subdivided into two main types:

1. endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination
   1. also includes replication errors
2. exogenous damage caused by external agents such as
   1. ultraviolet [UV 200-400 nm] radiation from the sun
   2. other radiation frequencies, including x-rays and gamma rays
   3. hydrolysis or thermal disruption
   4. certain plant toxins
   5. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents
   6. viruses^[5]

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).

Types of damage

There are several types of damage to DNA due to endogenous cellular processes:

1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species,
2. alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine, 6-O-Methylguanine
3. hydrolysis of bases, such as deamination, depurination, and depyrimidination.
4. "bulky adduct formation" (i.e., benzo[a]pyrene diol epoxide-dG adduct, aristolactam I-dA adduct)
5. 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.
6. Monoadduct damage cause by change in single nitrogenous base of DNA
7. Diadduct damage

Damage caused by exogenous agents comes in many forms. Some examples are:

1. UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage.
2. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.
3. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands. Low-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.
4. 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.^[6][7] 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.
5. 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.

Nuclear versus mitochondrial DNA damage

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 and apoptosis

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).^[8] 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,^[9] 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.^[10]

DNA damage and mutation

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damages and mutation are fundamentally different. Damages are physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damages can be recognized by enzymes, and, thus, they 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 damages and mutations are related because DNA damages often cause 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 damages are a special problem in non-dividing or slowly dividing cells, where unrepaired damages will tend to accumulate over time. On the other hand, in rapidly dividing cells, unrepaired DNA damages that do 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 damages in frequently dividing cells, because they give rise to mutations, are a prominent cause of cancer. In contrast, DNA damages in infrequently dividing cells are likely a prominent cause of aging.^[11]

DNA repair mechanisms

Single-strand and double-strand DNA damage

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.

Direct reversal

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.^[12] Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans,^[13] 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.^[14] 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.^[15] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

Single-strand damage

Structure of the base-excision repair enzyme uracil-DNA glycosylase. 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.^[14]

1. Base excision repair (BER) repairs damage to a single nitrogenous base by deploying enzymes called glycosylases.^[16] These enzymes remove a single nitrogenous base to create an apurinic or apyrimidnic site (AP site).^[16] 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.^[16]
2. 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 resysnthesis of removed DNA region.^[17] NER is a highly evolutionarily conserved repair mechanism and is used in nearly all eukaryotic and prokaryotic cells.^[17] In prokaryotes, NER is mediated by Uvr proteins.^[17] In eukaryotes, many more proteins are involved, although the general strategy is the same.^[17]
3. 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.^[18]

Double-strand breaks

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. Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination.^[14] 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]

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.^[19] 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.^{[20][21][22][23]} 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 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.^[24] 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.^[25]

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.

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.

A team of French researchers bombarded Deinococcus radiodurans to study the mechanism of double-strand break DNA repair in that organism. 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.^[26]

Translesion synthesis

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.^[27] 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.^[28] Paromita Raychaudhury and Ashis Basu^[29] studied the toxicity and mutagenesis of the same lesion in E.coli by replicating a G[8,5-Me]T-modified plasmid in Escherichia 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.^[30][31] 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.

Global response to DNA damage

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.^[32] 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.

DNA damage checkpoints

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,^[33] 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.^[34] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage.^[35] 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.^[36]

The prokaryotic SOS response

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.^[37] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes.^[38] 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.^[32] 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.^[38] 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.^[39] 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 transcriptional responses to DNA damage

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.^[40]

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.^[32]

DNA repair and aging

Pathological effects of poor DNA repair

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.^[11] 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.^[41] 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.^[42] 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.^[43]

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,^[11] 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.^[44]

Longevity and caloric restriction

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^[45] 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.^[46] 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.^[47] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents,^[48] although similar effects have not been observed in mitochondrial DNA.^[49]

It is interesting to note that 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.^[50] 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.

Medicine and DNA repair modulation

Hereditary DNA repair disorders

Defects in the NER mechanism are responsible for several genetic disorders, including:

• Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging
• Cockayne syndrome: hypersensitivity to UV and chemical agents
• Trichothiodystrophy: sensitive skin, brittle hair and nails

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

• Werner's syndrome: premature aging and retarded growth
• Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies (especially leukemias).
• Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents

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.

DNA repair and cancer

Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer.^[51][52] 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).

Epigenetic DNA repair defects in cancer

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.^[53]

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,^[54] changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1)^[55] 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.^{[56][57][58][59]}

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^[60][61] or in homologous recombinational repair (HRR).^[62] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.^[63]

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.^[64][65]

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).^[66] However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.^[67]

Frequencies of epimutations in DNA repair genes

A chart of common DNA damaging agents, the lesions they cause in DNA, the repair pathways utilized to repair these lesions, many of the genes in those pathways, indication of which genes are regulated epigenetically, and which of those epigenetically regulated genes are found with reduced 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).^[68] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.^{[69][70][71][72][73]}

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).^[74] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.^[75]

In further examples [tabulated in the article Epigenetics (see section "DNA repair epigenetics in cancer")], 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.^[76]

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.^[77] Of these, 83 are directly employed in the 5 types of DNA repair processes illustrated in the chart. The more well studied genes central to these repair processes are also shown in the chart. As indicated by the DNA repair genes shown in red, many of the genes in these repair pathways are regulated by epigenetic mechanisms, and these are frequently reduced or silent in various cancers (marked by an asterisk). Two review articles,^[59][78] and two broad experimental survey articles^[79][80] document most of these epigenetic DNA repair deficiencies.

It appears that epigenetic alterations in DNA repair genes are central to carcinogenesis.

DNA repair and evolution

The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophage (viruses that infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.^[81] 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.^[82]

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.

Rate of evolutionary change

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.^[83]