DNA damage response genes

Encyclopedia of Cancer, 2015


Definition

DNA damage response genes encompass all genes that encode proteins required for either direct or indirect response to DNA damage. The proteins encoded by these genes either act to help repair the damaged DNA within replicating cells or if damage is sufficient, to activate cell cycle checkpoints and if too severe, to activate cell death pathways. Each of the above enzymatic pathways also includes posttranslational modifiers such as protein kinases, phosphorylases, ubiquitin ligases, acetylators, sumoylators, methylators, scaffolding proteins required for proteasome complexes, and epigenetic gene silencing pathways. MicroRNA-mediated transcriptional regulation will likely be included in the future, as more information is gathered about this gene regulation system. The number of individual pathways and genetic sequences contributing to the major pathways above are unknown but likely encompass several 100 or more.

Characteristics

This essay will focus primarily on mutated genes associated with DNA damage response activities that have been identified as responsible for inherited autosomal recessive syndromes with increased incidence of human cancer (Table 1). These genes have been found mutated or epigenetically altered in sporadic tumors as well.

DNA replication polymerase alterations in cancer

DNA polymerases are essential for both replication and protection against genomic damage. Human cells contain at least 15 different DNA polymerases (Pols); Pols a, d, and e replicate chromosomal DNA in a 50 to 30 direction. High fidelity replication activity from these polymerases is essential for cellular viability. Pols d and e are responsible for the bulk of chromosomal replication and contain 30 to 50 exonuclease proofreading activity for removal of errors during replication. Pol a is responsible for initiating DNA synthesis on the leading strand and priming of Okazaki fragments during lagging strand DNA synthesis. These polymerases also perform fill-in synthesis during gap repair for specific DNA repair pathways, such as mismatch repair (MMR) and nucleotide excision repair (NER). Replicative DNA polymerases will not synthesize DNA over a damaged template, however. At least seven of the remaining mammalian DNA polymerases now have defined activity as translesion synthesis (TLS) polymerases. The TLS Pols have decreased fidelity of replication with no proofreading exonuclease activity. The function of these Pols is putatively to avoid replicating polymerases stalling before unrepaired template lesions at the fragile replication fork, which would otherwise cause DNA strand breaks and genomic instability. Each of the TLS Pols possesses a unique type of DNA damage bypass activity. The only DNA polymerase for which an inherited deficiency, in the form of a disabling mutation, predisposes humans to cancer is the TLS DNA Polymerase DNA Pol h and is one of several different mutated genes that result in xeroderma pigmentosum (XP). XP is an inherited disorder with eight complementation groups (see below), one of which is XPV – a lack of DNA Pol h activity. This disorder is associated with up to a 1,000-fold increased risk for sunlight-induced skin cancers. Pol h synthesizes correctly over a DNA template containing TT-cyclopyrimidine dimers (CPDs), the most common lesion resulting from ultraviolet (UV) light exposure. Surprisingly, whole genome sequencing of thousands of genomes from several different types of cancers has yet to reveal specific cancer-associated alterations in any other DNA polymerase gene, nor has gene silencing been observed by epigenetic mechanisms. Several different mouse models have been developed that express different DNA Pol mutations that are predisposed to various tumors, however.

Table 1. Human hereditary cancer syndromes due to autosomal recessive inherited defects in DNA damage response genes

Syndrome Gene Cancer Disabled process
Xeroderma pigmentosum XPA а XPG, XPV UV-induced skin cancer NER, TLS
Hereditary nonpolyposis colon cancer MSH2, MLH1, MSH6, PMS2 Colon cancer MMR, MMR-induced DNA damage signaling
MYH-associated polyposis MYH Colon cancer BER
Hereditary breast and ovarian cancer BRCA1, BRCA2 Breast, ovarian cancer HR
Fanconi anemia FANCA а FANCP, RAD51C Leukemia DNA cross-link repair
Ataxia-telangiectasia ATM Leukemia, lymphoma, breast cancer DNA DSB-induced damage signaling, G2 checkpoint
Nijmegen break syndrome NBS Leukemia, lymphoma DNA DSB repair
Seckel syndrome ATR Acute myeloid leukemia DNA SSB-induced damage signaling, G2 checkpoint
Li-Fraumeni TP53 Multiple cancers DNA damage signaling, G1 checkpoint
Werner syndrome WRN Multiple cancers Helicase and exonuclease functions
Bloom syndrome BLM Multiple cancers Helicase functions, HR
Rothmund-Thomson RECQL4 Osteosarcoma, skin cancer Helicase functions ?

DNA repair pathway alterations in cancer

Chromosomal DNA is susceptible to errors by DNA replication machinery during every cell cycle in addition to constant attack by mutagens produced by endogenous metabolism as well as exogenous sources. DNA repair machinery must be able to correctly repair such damage quickly and correctly in replicating cells or undergo the risk of mutations that alter cellular phenotype. Cancer genome sequencing has confirmed that cancer cells bear up to 100,000 more mutational events than found within the genomes of normal cells. However, differences in mutation prevalence between individual cancers and the vast number of these mutations reveal considerable information about the development of neoplasia. The somatic mutation signatures of individual tumors often carry imprints characteristic of mutagenic exposure or DNA repair deficiencies. For example, the mutational pattern in skin cancer reveals overwhelming exposure to UV light or tobacco carcinogens in lung cancer or an inherited deficiency of DNA mismatch repair in colorectal cancer.

Development of carcinogenesis during clonal expansion requires a subset of “driver mutations” to fall within a key set of “cancer genes.” These mutations confer abilities for clonal growth, invasion, and metastasis, as well as impairment of programmed cell death or senescence pathways. The vast majority of “passenger” mutations, by definition, do not confer growth advantage. The increased mutational load within neoplastic cells also indicates that cancer is a process requiring many cell cycles and that the mutation rate in cancer cells is much higher than in normal cells; therefore, a malfunctioning DNA repair system is a required “driver” mutation at some point during carcinogenesis. Several inherited autosomal recessive cancer syndromes (mutated gene in germ cells) demonstrate the importance of DNA repair for normal growth and division of somatic cells. For example, XP is a complex genetic disorder that places a person at greatly increased risk of sunlight-induced skin cancer (see above). Seven of the eight complementation groups discovered in this inherited syndrome are due to disabling mutations within the NER pathway (XPA-G). This DNA repair pathway is required to repair bulky DNA damage inflicted by exogenous agents such as CPDs produced by UV light from the sun. Indeed, the mutational signature in XP cells clearly derives from unrepaired CPD lesions.

Hereditary nonpolyposis colon cancer (HNPCC) is a familial cancer syndrome representing 2–3% of all colon cancer cases. This cancer syndrome is due to germ line mutations in DNA mismatch repair genes, most often hMSH2 or hMLH1. Genomic instability within cancer cells from this inherited syndrome is of a strikingly different pattern than the majority of tumors. Most human tumors display increased chromosomal instability (CIS) with many types of large chromosomal aberrations, such as large insertions, deletions, or translocations evident by cytogenetic analyses. HNPCC cells instead display microsatellite instability (MSI) evident only by sequencing of specific genomic locations. Microsatellite sequences are regions of homopolymeric stretches of nucleotides that replicating DNA polymerases have more difficulty synthesizing through, requiring frequent corrective input by the MMR system. Without this backup repair pathway, each replication cycle leaves more contraction and expansion errors within these sequences by the “slipping and sliding” errors made by the replicating polymerases. Many driver mutations in MMR defective tumors are within genes having microsatellite repeats nested in their sequences. Approximately 15 % of sporadic gastric, colorectal, and endometrial tumors also exhibit MSI rather than CSI. The majority of these sporadic tumors have defective MMR because of epigenetic silencing via promoter hypermethylation of hMLH1.

An additional function of the MMR system, less well understood than DNA mismatch repair, is the ability of MMR proteins to recognize and bind to specific types of DNA damage, such as O6methyldeoxyguanine (O6meG), as a signal for the DNA damage response system to initiate cell cycle arrest either for subsequent repair or apoptosis if the DNA damage cannot be repaired. Monofunctional alkylating agents, such as N-methyl-N0-nitro-N-nitrosoguanidine (MNNG) or the clinical equivalent temozolomide (TMZ), produce several different alkylated DNA adducts in addition to O6meG, the majority of which have low mutagenic potential and are repaired efficiently by the base excision repair pathway (BER). The O6meG modification, however, is not repaired by BER but instead by a one-step enzymatic reaction that directly and covalently transfers the methyl group from the O6meG position to methylguanine methyltransferase (MGMT), thus rendering this enzyme useless for further reactions and earning its alternate name as the “suicide enzyme.” MGMT can be rapidly depleted in the face of highly alkylated DNA. As well, MGMT is frequently expressed at low levels or epigenetically silenced in different tissues (normal and malignant). Low or absent MGMT expression in tumor cells can be important if alkylation chemotherapeutics, such as TMZ, are to be effectively used. For example, if the cell undergoes DNA replication before repair of O6meG can occur, because of low or absent MGMT, there is an elevated likelihood of misinsertion of thymine opposite the damaged guanine leading to increased GаA transition mutations. A sufficient level of O6meG within chromosomal DNA, however, will trigger an MMR-induced DNA damage response and subsequent apoptosis. To further complicate matters, both TLS and homologous recombination (see below) have been strongly implicated to also have roles in the DNA damage response to O6meG. Conversely, cells deficient in both MMR and MGMT expression do not trigger a DNA damage response or undergo apoptosis. MMR- and MGMT-deficient cells therefore demonstrate a significantly increased mutation rate, because of increased cell survival as well as lack of both mismatch and O6meG repair within cells exposed to alkylating agents.

The base excision repair pathway (BER) removes small DNA lesions and mistakes that are frequently the result of endogenous damaging events, such as alkylation or oxidation damage, or abasic sites. BER has not been found to have inherited deficiencies in function that significantly increase susceptibility to cancer, with the exception of MYH, a glycosylase that removes misinserted adenines opposite 8-oxo-deoxyguanine as a repair step during oxidative damage repair. MYH-associated polyposis (MAP) is an inherited colon cancer syndrome resulting from disabling mutations within the MYH gene. The BER pathway has come under increasing scrutiny recently because of its major role in the repair of chemotherapeutic alkylation DNA damage. There are now numerous mouse models with altered BER protein expression that exhibit increased or decreased susceptibility to alkylation-induced cancer. Specific inhibitors of particular BER enzymes have been shown to potentiate the toxicity of clinical alkylators such as TMZ.

Perhaps the most highly recognized inherited defects in DNA repair within the lay public are those associated with BRCA1 and BRCA2 genes, conferring an increased susceptibility to breast and ovarian cancer. BRCA1 (but not BRCA2) is also frequently found epigenetically silenced in various sporadic cancers. These gene products are part of the homologous recombination (HR) pathway for repair of DNA double-strand breaks that is active in late S and G2 phases of the cell cycle. Tumors lacking expression of BRCA1 or BRCA2, thus lacking a functional HR pathway, have created recent excitement as a target for “synthetic lethal” approaches to chemotherapy. Tumor cells that lack one DNA repair pathway are often highly susceptible to inhibitors of other DNA repair pathways used by the cell to compensate for the deficient pathway. For example, inhibitors to Poly ADP-ribose polymerase (PARP) effectively inhibit BER and have been discovered to be highly toxic to cells also lacking HR repair when combined with DNA damaging chemotherapy or radiation therapy. This type of synthetic lethal approach is proving effective for TMZ-induced alkylation damage as well. The principal idea is to increase nonrepairable DNA damage in the tumor cells to unsustainable levels in a very targeted approach, using the cell’s own genetic deficiency as a chemotherapy-directed tool against itself. There is now great excitement in the field as more synthetic lethal approaches are sought within other DNA repair pathways.

Fanconi anemia (FA) has up to 15 genes now associated with this inherited disorder (FANCA-P, RAD51C). Disabling mutations in any of these genes results in an inability to repair DNA damaged by cross-linking agents and a highly increased susceptibility to acute myelogenous leukemia. The complex FA pathway coordinates NER, HR, and TLS pathways to resolve interstrand cross-links during replication. There is little knowledge in regard to epigenetic inactivation of this gene family in cancer, although some cases involving epigenetic inactivation of FancF have been reported.

DNA damage signaling pathways in cancer

Gene products such as ATM, ATR, and p53 are frontline defenses that initiate cell cycle checkpoint arrest at specific parts of the cell cycle for repair of DNA damage, or if damage is too severe, these gene products trigger apoptosis.

Ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) are both inherited disorders with increased sensitivity to ionizing radiation and other treatments inducing DNA double-strand breaks. Both syndromes have increased risk of several different lymphatic tumors. ATM (ataxia-telangiectasia mutated) is the mutated gene that is inherited in AT. AT individuals also have an increased risk for breast cancer that has been ascribed to ATM’s interaction and phosphorylation of BRCA1 following DNA damage. ATM is a protein kinase that normally initiates G2 arrest after DNA double-strand breaks occur. In Nijmegen breakage syndrome, NBS is the inherited gene that is mutated. NBS codes for nibrin (NBS1), a protein that is normally part of a trimeric complex (NBS1/MRE11/RAD50) responsible for coordinating repair of DNA double-strand breaks and recruiting ATM to the strand breaks to initiate cell cycle arrest.

ATR (ATM and RAD3 related) initiates G2 cell cycle arrest in response to persistent single-strand DNA breaks. ATR is required for cell viability, although certain inherited mutations have been found to be responsible for Seckel syndrome, which is an autosomal recessive disorder characterized by dwarfism, intrauterine growth retardation, bird-like facies, microcephaly, and mental retardation. ATR-Seckel syndrome has been found in patients with mutations in ATR. Cell lines from patients with Seckel syndrome, who are normal for ATR, show defective ATR signaling, suggesting that Seckel syndrome can be caused by mutations in other components of the ATR pathway.

Li-Fraumeni syndrome is the inherited deficiency of p53 due to germ line mutations in the gene TP53. Several different types of tumors are associated with this syndrome. The TP53 gene is also mutated in approximately 50 % of all sporadic human tumors. Cells expressing mutated p53 do not exhibit G1 cell cycle arrest, nor do they undergo p53-dependent apoptosis.

Werner syndrome presents as an accelerated aging process. Increased telomere attrition and genomic instability is observed in this syndrome and in sporadic tumors that express the dysfunctional protein from this mutated gene. The WRN gene responsible for this syndrome codes for a RECQ helicase family member (RECQL2). The WRN protein has both helicase and exonuclease functions that participate in many aspects of DNA metabolism, including maintenance of telomere structure and homology-dependent recombination. This gene has also been reported as epigenetically silenced in a large number of sporadic tumors.

Bloom syndrome is due to mutations of the BLM gene, which belongs to the DExH box-containing RecQ helicase subfamily. Cells harboring this mutated gene have a spontaneous mutation rate ten times higher than normal with highly increased rates of chromosome breakage and excessive HR activity. This helicase protein is thought to play a major role in DNA replication and HR.

Rothmund-Thomson syndrome is a very rare disorder that can severely affect many parts of the body, with increased incidence of osteosarcoma and skin cancer predominating. The RECQL4 helicase gene is mutated in this disorder. The gene product appears to play some role in replicating and repairing DNA, although little is understood currently about its function.

References

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