Recall that on the order of 105 DNA lesions are produced per cell per day (Sec. 5.2.1). Cells have evolved multiple mechanisms to detect and remove these lesions to prevent propagation to progenitor cells and, in the case of multicellular organisms, suppress tumorigenesis. An improved understanding of the mechanisms of DNA repair came from the isolation of repair-deficient rodent cells (ie, Chinese hamster ovary [CHO] mutants) with unusual sensitivity to different classes of DNA-damaging agents. Some mutants exhibited extreme sensitivity to UV light and crosslinking agents, such as mitomycin C, but little or no sensitivity to x-rays. Other cells exhibited sensitivity to x-rays and chemical agents known to cause DNA breakage, but little or no sensitivity to UV light or crosslinking agents. These various phenotypes, which are similar to those characterized previously in bacteria and yeast, indicate the involvement of several distinct DNA repair pathways and associated gene products. Some of these repair pathways are so highly conserved from yeast to humans that yeast proteins can substitute for human proteins and vice versa; this has been helpful in the cloning and functional characterization of the human homologs of yeast DNA repair genes.
Some frequent lesions, such as those formed by oxidation or DNA-reactive carcinogens, induce structurally distinct mutagenic and cytotoxic damage to the DNA (see Chap. 4, Sec. 4.3). Some of these adducts are recognized and repaired by a class of enzymes that are used only once. For example, induction of O(6)-methylguanine (O(6)-MeGua) by oxidation or N-nitroso compounds is recognized by O(6)-alkylguanine DNA alkyltransferase, which reverts the O(6)-MeGua back to guanine in a single-step irreversible reaction that inactivates the enzyme and prevents mutagenic G:C→A:T transitions.
An important property in DNA repair is the fidelity of the repair pathway leading to the concepts of error-prone, and error-resistant (or error-free), DNA repair. Many DNA lesions can block transcription of RNA, thereby inactivating the DNA damage-containing gene on the DNA strand that is being transcribed. Persistent blockage of RNA synthesis can lead to cell death so these lesions are often repaired through the transcription-coupled repair pathway (see Sec. 5.3.3 below); this pathway is designed to displace the stalled RNA polymerase and drive a high-priority repair mechanism. For lesions that block progression of the replication fork during DNA replication, several error-prone DNA polymerases have been described that have increased flexibility and low fidelity to allow for replicative bypass (ie, translesion DNA synthesis) of the base damage contained within DNA. These polymerases can be used temporarily by the cell during acute DNA replication damage and substituted by more accurate DNA polymerases at a later time. Use of these lower-fidelity bypass DNA polymerases can contribute to high error-rates during DNA replication and may lead to malignant transformation. This process is diagrammed in Figure 5–3 and is known as translesion DNA synthesis.
Translesion DNA synthesis. The "DNA polymerase switch model" for translesion synthesis and mutagenesis. This cartoon shows the replicative DNA polymerase (yellow sphere) blocked at a template lesion site. Cells contain several different DNA polymerases (other indicated spheres) that transiently replace the replicative DNA polymerase. After a short patch of synthesis in the vicinity of the lesion, the replicative DNA polymerase resumes high fidelity and processive synthesis. (Adapted from Cordonnier and Fuchs, 1999.)
In the following sections, discrete biochemical pathways of DNA repair are described. These pathways can be divided into different classes depending on the specific DNA lesion they are designed to repair, and include (a) mismatch repair, (b) base excision repair, (c) nucleotide excision repair, (d) single-strand break repair, and (e) homologous and nonhomologous repair of DNA double-strand breaks.
The mismatch repair (MMR) pathway is enacted when the DNA-polymerase inserts an incorrect base during DNA replication or when the polymerase creates helical distortions by inserting or deleting bases in short oligonucleotide repeats (microsatellites) during replication. These helical distortions are termed insertion–deletion loops. The protein products of MMR genes form heterodimer complexes, and different protein pairs recognize specific mismatched nucleotides or insertion-deletion loops in DNA (Fig. 5–4). For example, the MSH2 protein forms a heterodimer with an additional MMR protein, MSH6 or MSH3, and the resulting complexes are called MutS-α or MutS-β, respectively. MUTS-α is required for the recognition of DNA base–base mismatches, whereas MutS-α and MutS-β have partially redundant functions for the recognition of DNA insertion–deletion loops. A second heterodimer forms between the MMR gene product MLH1 and PMS2 or MLH3 to form MutLα and MutLβ, respectively. The MutL complexes coordinate the interplay between the initial mismatch recognition complex and subsequent protein interactions required to complete MMR. The latter proteins include proliferating cell nuclear antigen (PCNA), DNA polymerases δ and ε, and possibly DNA helicases that unwind the DNA helix to facilitate DNA synthesis (Hoeijmakers, 2001). These processes are diagrammed in Figure 5–4.
Mismatch repair (MMR). The mismatch repair pathway is initiated either when a base is misincorporated to create a base–base mismatch (BBMM) or when insertion–deletion loops (IDLs) are created during replication in microsatellite regions. These lesions are recognized by different complexes: MSH2/MSH6 (MutSα) + MLH1/PMS2 (MutLα) recognize BBMMs and IDLs, whereas MSH2/MSH3 (MutSβ) + MutLα or MLH1/MLH3 (MutLβ) can also recognize IDLs. Whichever MutS/L complex is utilized, it translocates along DNA until it reaches the proliferating cell nuclear antigen (PCNA) associated with the replication factor complex (RFC) at the site of the replication fork. Next, the nuclease EXO1 is loaded and activated, which resects the fork backward past the lesion, at which point MutS/L and EXO1 are unloaded. The DNA polymerase δ and ε(POL δ/ε) fills the gap and Ligase I seals the nick if necessary. (Adapted from Jiricny, 2006.)
5.3.2 Base Excision Repair
Spontaneous oxidative damage is known to occur in cells producing 104 to 105 oxidative residues, such as 8-oxo-deoxyguanosine, per cell per day among the approximately 3 × 109 bases in the genome. DNA base damage, occurring as a result of endogenous oxidative processes or exogenous DNA damage (eg, from ionizing radiation) is repaired by the base excision repair pathway. Base excision repair involves the enzymatic removal of the damaged DNA base by DNA glycosylases. DNA glycosylases are a family of enzymes that cleave glycosidic bonds and are specific to particular base lesions. There are 2 classes of DNA glycosylases that differ in their reaction mechanism: monofunctional enzymes leave the DNA strand intact and bifunctional DNA glycosylases also cleave the DNA backbone (Fig. 5–5). For example, the OGG1 protein is a bifunctional 8-oxoguanine DNA glycosylase that removes spontaneous or ionizing radiation-induced lesions to prevent cellular mutations. During base excision repair the initial base removal step leaves an apurinic or apyrimidinic site that is similar to single-strand DNA breaks. Such single-strand breaks can be induced by free radicals or by ionizing radiation without the action of DNA glycosylases, and repair of these 2 types of lesions (base damage and single-strand breaks) converge into this common pathway. Base excision repair and single-strand break repair involve similar components and the processes are shown in Figure 5–5. The major pathway is short-patch base excision repair and involves the replacement of a single nucleotide following DNA backbone cleavage at the base excision site. A minor pathway is the long-patch base excision-repair pathway, which exists for the repair of 2 to 13 damaged nucleotides.
Mechanism of base excision repair (BER) and single-strand break repair (SSBR). Damaged bases are removed by the BER pathway and single-strand breaks are repaired by components of the same pathway. There are 2 subpathways, known as short- and long-patch BER, and pathway choice depends on the initial glycosylase enzyme that is required to recognize and remove the particular base lesion. When the enzyme includes β-lyase activity, the base is removed, leaving an apyrimidinic (AP)-site with a nick in the DNA backbone and a flap 3′ to the removed base. The APE1 endonuclease removes this flap and the POLβ enzyme fills in the gap. Simultaneously the single-stranded DNA recruits the poly(ADP) ribose polymerase (PARP) enzyme that facilitates recruitment of ligase III and XRCC1 that seal the nick. Because only a single nucleotide is replaced, this is called short-patch BER. In the complementary long-patch BER pathway the recognition glycosylase does not contain β-lyase activity and leaves an AP site with an intact DNA backbone. The APE1 endonuclease catalyses the formation of a flap 5′ to the removed base, and PCNA, POL δ/ε, and replication factor C (RFC) initiate synthesis and create a longer flap of 2 to 10 nucleotides. The FEN1 nuclease cleaves the overhang and the nick is sealed by ligase I. (Adapted from Sancar et al, 2004.)
No human disorders have been related directly to inherited deficiencies in base excision repair, and knockout mice engineered to lack core proteins in the pathway die as embryos, attesting to its important role in development. Genetic mouse knockout models for a variety of glycosylase genes have shown only mild increases in genetic mutations. This may be because of partial redundancy in the glycosylases and/or overlap with the transcription-coupled repair processes described below. However, base excision repair may be defective in cells that have mutations in p53 as the p53 protein can stimulate base excision repair by direct interactions with APE1 and DNA-Polβ (Offer et al, 2001). Indeed, the gene locus encoding the glycosylase 8-Oxoguanine glycosylase (OGG1) on chromosome 3p25-26 is frequently lost in lung cancers, consistent with a purported role in preventing carcinogenesis.
An exciting area of exploration is the clinical utility of poly(ADP)-ribose polymerase (PARP) inhibitors. As shown in Figure 5–5, PARP activity is required as an intermediate step preceding DNA synthesis during base excision repair or when single-stranded DNA regions are recognized. When PARP is inhibited by small molecule inhibitors, this causes the accumulation of single-stranded gaps that can cause collapse of replication forks during DNA replication. Under normal circumstances homologous recombination (see below) during DNA replication can rescue this and prevent double-strand break (DSB) formation. However, when homologous recombination is defective, such as in BRCA2-deficient breast cancers, these PARP inhibitors can lead to accumulation of DSBs and cell death (Helleday, 2010). This general concept of inhibiting compensatory pathways in tumor cells, while sparing normal tissues, is termed synthetic lethality, a common concept in yeast biology, and it has become an exciting area of research for novel cancer therapies (see Chap. 17, Sec. 17.3.2).
5.3.3 Nucleotide Excision Repair
In aqueous solution, DNA is susceptible to absorption of photons in the range of 200 to 300 nm, which increases reactivity of pyrimidine bases to produce 6-4 photoproducts (6-4PPs) and interstrand crosslinks in the form of cyclobutane pyrimidine dimers (CPDs; eg, thymine–thymine linkages). These lesions, and other bulky chemical adducts in DNA, are removed by nucleotide excision repair (NER), which is a complex DNA repair pathway involving more than 30 genes. Many of the NER genes were originally cloned from complementation analyses of cells from patients with XP and with cells from patients with Cockayne syndrome (CS), and are referred to as XPA-XPG or CSA or CSB in protein nomenclature. Patients with these syndromes have severe sensitivity to UV light and a much higher incidence of skin cancer (Fig. 5–6).
NER and XP. Left: Incidence of skin cancer in people with XP compared to the normal population with similar exposure to the sun. The 1000-fold excess risk of UV-induced skin cancer is secondary to defective repair of UV-induced DNA lesions in XP patients. Right: NER proceeds by initial recognition of the lesion by 1 of 2 different complexes, depending on whether "global genome"-nucleotide excision repair (GG-NER) or "transcription-coupled" repair (TCR) is used. In GG-NER helical distortions stimulate recognition by the xeroderma pigmentosum complementation group C (XPC)-hHR23B complex. Subsequently, XPA is recruited and transcription factor IIH (TFIIH) causes formation of an "open complex." The single-stranded regions of DNA are recognized by RPA. Two endonucleases, XPG and XPF/ERCC1, cut 3′ and 5′ to the lesion, respectively. The approximately 30-nucleotide gap is filled by POL δ/ε and the nick is sealed by ligase I. In TCR, the RNA polymerase II transcription machinery is thought to facilitate recognition of the lesion that, along with the CS proteins A and B (CSA/B), contribute to recognition of the lesion and induction of NER in a similar mechanism to GG-NER. The entire process of NER takes several minutes to complete.
The process of NER is highly conserved in eukaryotes and consists of the following 4 steps: (a) recognition of the damaged DNA; (b) excision of an oligonucleotide of 24 to 32 residues containing the damaged DNA by dual incision of the damaged strand on each side of the lesion; (c) filling in of the resulting gap by DNA polymerase; and (d) ligation of the nick (Balajee and Bohr, 2000). In human cells, NER requires at least 6 core protein complexes for recognition of damage and dual incision (XPA, XPC-hHR23B (human homolog of RAD23B), RPA (replication protein A), TFIIH (transcription factor IIH), XPG and ERCC1-XPF (excision repair cross complementation 1) and other factors for DNA synthesis and ligation to complete repair (PCNA, RFC [replication factor C], DNA polymerase δ or α, and DNA ligase I) (de Laat et al, 1999). The process of NER is diagrammed in Figure 5–6.
NER consists of 2 subpathways that differ in their mode of recognition of the helical distortions that the lesions produce. The first subpathway, termed global genome repair (GG-NER) is transcription-independent and surveys the entire genome for DNA lesions. The 6-4PPs, which distort the DNA more than CPDs, are removed rapidly, through recognition by the XPC-HH23B protein complex in GG-NER. In contrast, CPDs are repaired very slowly by GG-NER and are removed more efficiently from the transcribed strand of expressed genes by transcription-coupled repair (TCR). During TCR, the stalled RNA polymerase induces the recognition of the DNA lesions on the transcribed strand, and this process is facilitated by CSA and CSB proteins (Friedberg, 2001). The TCR and GG-NER pathways converge after this recognition step to form the open complex and removal of the damage as shown in Figure 5–6.
5.3.4 DNA Double-Strand Break Repair: Homologous Recombination
DSBs in DNA result from ionizing radiation and certain chemotherapeutic drugs, from endogenously generated reactive oxygen species, and from mechanical stress on the chromosomes (Zhou et al, 1998). They can also be produced when DNA replication forks encounter DNA single-strand breaks, following defective replication of chromosome ends (ie, telomeres, see Sec. 5.6) or when topoisomerase enzymes are inhibited (eg, by etoposide) preventing the rejoining of the DSBs these enzymes induce. In addition, DNA DSBs are generated to initiate recombination between homologous chromosomes during meiosis and occur as intermediates during developmentally-regulated rearrangements, such as V(D)J recombination during the generation of immunoglobulins (see Chap. 21, Sec. 21.3.1).
In human cells, repair of DNA DSBs occurs either by homologous recombination (HR; Fig. 5–7) or nonhomologous end-joining (NHEJ; Fig. 5–8). The preferred pathway depends on tissue type, the extent of DNA damage, the cell-cycle phase in which the cell is damaged, and the relative need for repair fidelity. There may also be cooperation between the 2 pathways (Richardson and Jasin, 2000). Repair by HR requires homology between the broken DNA strand and the template strand used in repair. Typically, this is newly replicated sister chromatid and as a result HR is restricted to the S and G2 cell-cycle phases (see Fig. 5–7). The HR pathway results in error-resistant repair of DNA DSBs because the intact undamaged template is used to pair new DNA bases between the damaged and undamaged strands during DNA synthesis.
HR of DNA DSBs. HR is a major pathway for DSB repair and requires the presence of a sister chromatid. Here, single-stranded DNA overhangs are created and stabilized by nucleases (MRN [MRE11-RAD50-NBS1] complex, CtIP, EXOI, DNA2), helicases (BLM, WRN), and the single-strand DNA binding protein RPA. These RPA-coated overhangs are then exchanged to create RAD51-coated overhangs, a process facilitated by the RAD52 group of proteins (RAD52, RAD51B/C/D, XRCC2/3), BRCA2, and RAD54B, that also contributes to scanning for homologous DNA regions. This RAD51-filament can then invade the complementary DNA-duplex to form a D-loop. DNA polymerases then extend this invading strand and the corresponding strand on the opposite side of the DSB. (Adapted from Helleday et al, 2007.)
NHEJ of DNA DSBs. NHEJ is composed of subpathways that repair the DNA lesion. In the "fast" NHEJ pathway, the KU70/80 heterodimer recognizes the DNA ends and recruits the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), which autophosphorylates and forms the active DNA-PK complex. Downstream the ligase IV/XRCC4 dimer associates with the XLF/Cernuous proteins and joins the DNA ends together. In some cases, the DSB ends at the break are unligatable and require processing in the so-called slow NHEJ pathway. Repair of these ends depends at least in part on the ATM kinase and is processed by nucleases such as MRE11 or Artemis, kinases or phosphatases like PNKP, or filled in by Polμ/λ. Once competent for ligation, these ends follow the same XRCC4/ligase IV pathway above. Some lesions may require chromatin modification by KAP-1 for repair.
A less-well understood parallel pathway is microhomology-mediated end-joining (MMEJ)/single-strand annealing (SSA), which can be thought of as a hybrid pathway for DSB repair. The distinguishing features of MMEJ and SSA are poorly defined, but are differentiated primarily by the extent of the homologous regions involved and the requirement of RAD52 for SSA in yeast. The initial step includes end resection to expose homologous regions by nucleases, including MRE11, CtIP, EXO1, and BRCA2. The annealing of the homologous single-strand overhangs is facilitated by the RAD51 protein and the remaining flaps are trimmed by XPF-ERCC1. The involvement of polymerases and the PARP enzyme in this step is likely necessary but unknown. The nicks are sealed with ligase I or IIIα. Despite the disadvantage of its low fidelity, this pathway can act quickly, as required of an emergency mechanism, and, unlike HR, it does not depend on sister DNA molecules, which exist in the cells only after DNA replication. (Adapted from Dobbs et al, 2010; McVey and Lee, 2008.)
The HR pathway is highly conserved, likely owing to its ability to maintain the integrity of genetic information. Shown in Figure 5–7 is a general schematic of HR processes where a DSB is initially recognized by the MRN complex (MRE11-RAD50-NBS1). MRE11 possesses both single-stranded DNA (ssDNA) endonuclease and 3′ to 5′ exonuclease activities, which are enhanced by CtIP. Together with the 5′ to 3′ exonuclease EXO1 and endonuclease DNA2, these proteins collaborate with helicases (ie, BLM and WRN) to form resected ssDNA, which is rapidly coated with replication protein A (RPA). Exchange of RPA for the RAD51 protein is facilitated by the RAD52 epistasis group, XRCC2/3, BRCA1/2, and RAD54B, which also contributes to strand invasion of the sister chromatid forming a D-loop (Heyer et al, 2010; Svendsen and Harper, 2010). DNA synthesis extends the invading strand and the D-loop is resolved by poorly understood mechanisms. There are actually several subpathways of HR that vary in their ability to prevent crossover of information from the strand acting as the template for DNA synthesis during the formation of so-called Holliday junctions (see Fig. 5–7; Helleday et al, 2007; Heyer et al, 2010).
The BRCA1/2 breast cancer-susceptibility proteins (see also Chap. 7, Sec. 7.6.3) also play a role in the homologous repair of DNA DSBs. Both BRCA1 and BRCA2 proteins form discrete nuclear foci during S-phase following exposure to DNA damaging agents at the sites of DNA damage. Although RAD51 colocalizes at subnuclear sites with BRCA1, their interaction is thought to be indirect, with only 1% to 5% of BRCA1 in somatic cells associating with RAD51 (Marmorstein et al, 1998). In contrast, the BRCA2 protein contains 8 BRC repeats, each of 30 to 40 residues, which are the major sites for the direct binding to RAD51 by a substantial fraction of the total intracellular pool of BRCA2 (Davies et al, 2001). As such, BRCA2-deficient cells have 10-fold lower levels of HR when compared to BRCA2-proficient cells (Moynahan et al, 2001). One model suggests that a BRCA2-RAD51 complex promotes the accurate assembly of DNA repair proteins required to offset DNA breaks that accumulate during DNA replication; these could otherwise lead to gross chromosomal rearrangements, LOH at tumor-suppressor gene loci, and carcinogenesis. In some instances, when a single-stranded gap is met by the replication machinery, a single DSB will be produced, which utilizes HR as a mechanism to restart replication forks if the delay is not prolonged.
Biochemical and genetic studies in yeast have been fundamental in the ability to clone human homologs of proteins involved in the HR pathway. In the yeast, Saccharomyces cerevisiae, the RAD52 group of genes are involved in HR including RAD50, RAD51, RAD52, RAD54B, RAD55, RAD57, RAD59, MRE11, and XRS2 (the latter retermed p95 or NBS1-nibrin in mammalian cells). RAD51–/– mice are embryonic lethal, attesting to the importance of this critical HR protein in meiosis and development. Careful observations during the initial stages of embryogenesis in RAD51–/– mice show that lethality is preceded by chromosomal rearrangements and deletions. It is thought that DNA replication errors and replication-associated DNA strand breaks are converted into DNA DSBs in HR-defective cells (Lim and Hasty, 1996). In cells derived from RAD54–/– mice (which are developmentally normal), there is also decreased HR and increased hypersensitivity to DNA crosslinking agents such as mitomycin C (Essers et al, 1997). Although in S. cerevisiae RAD52 is essential for DNA DSB repair, RAD52–/– mice are viable and fertile and do not show a DNA DSB repair deficiency. HR therefore appears to be more complex in mammalian cells than in yeast, possibly as a result of functional redundancy in many of the proteins.
A number of human cancers, including ovarian, breast, prostate, and pancreatic, have mutations or altered expression and function of the MRE11, RAD51/RAD52/RAD54, and BRCA1/2 genes. This observation suggests that tumorigenesis is associated with altered HR in sporadic tumors. Increased levels of RAD51 expression in certain cancer cell lines also has been associated with altered phosphorylation, ubiquitination and transcription of the RAD51 protein as a result of abnormal c-ABL– and STAT5-mediated tyrosine kinase signaling pathways in cancer cell lines (see Chap. 8). This can lead to acquired radioresistance and chemoresistance (Daboussi et al, 2002).
5.3.5 DNA Double-Strand Break Repair: Nonhomologous End-Joining
The NHEJ pathway is outlined in Figure 5–8. The recognition step of NHEJ is initiated by high-affinity binding of the KU70/80 heterodimer to the DNA ends, which causes a conformational change that recruits DNA-dependent protein-kinase catalytic subunit (DNA-PKcs). Autophosphorylation of DNA-PKcs at multiple sites is essential for NHEJ to occur and appears to mediate DNA-PKcs dissociation from the break site (Dobbs et al, 2010). Many DNA DSBs have damaged ends that need to be processed to restore their ability to be ligated; for example, if 5′-phosphates are not present and/or 3′-phosphate groups need to be removed to create blunt ends. These structures are repaired by "end processors" that are partially dependent on the activity of the kinase mutated in ataxia telangiectasia (ATM [ataxia-telangiectasia mutated]) and include the Artemis protein, CtIP and MRN complexes, which are nucleases, PNKP which is both a 5′ kinase and 3′-phosphatase and Polμ/λ. Once ends are restored the XRCC4-ligase IV complex, stimulated by XRCC4-like factor (XLF), rejoins the ends (Hiom, 2010). MMEJ and single-strand annealing (SSA), are minor subpathways of NHEJ that require end resection to reveal short (5 to 25) and longer (>30) homologous stretches of DNA, respectively (see Fig. 5–8). Although this pathway is much less understood, it is believed to contribute to deletions and chromosomal rearrangements that result in genomic instability (McVey and Lee, 2008).
The major protein complexes implicated in the NHEJ pathway are the DNA-dependent protein kinase (DNA-PK) complex and the XRCC4/ligase IV complex. Human DNA-PK consists of an approximately 460-kDa DNA-PK catalytic subunit (DNA-PKcs), and a DNA end-binding KU heterodimer (consisting of 70-kDa and 80-kDa protein subunits). The catalytic subunit shows homology to the phophatidylinositol-3 kinase (PI3K) superfamily at its C-terminus, which contains the protein kinase domain required for phosphorylating DNA-PK–associated proteins during repair. Mutations in either DNA-PKcs or in one of the KU genes result in sensitivity to ionizing radiation and reduced ability to repair radiation-induced DNA DSBs. XRCC4 forms a stable complex with DNA ligase IV and XLF, and probably links detection of the initial lesion by DNA-PK to the actual ligation reaction carried out by ligase IV (see Fig. 5–8) (Pang et al, 1997).
Much information regarding the cellular activity of the DNA-PK complex stems from research utilizing the severe combined immunodeficiency (SCID) model mouse, which has a complete lack of mature T and B cells and is radiosensitive. The DNA-PKcs protein in the SCID mouse is mutant and unstable because of a loss of the last 83 amino acids prior to the C-terminal kinase domain. Consequently, DNA-PK activity is severely reduced in tissues derived from this animal. Immunodeficiency is secondary to an inability to process and rejoin the broken DNA molecules produced endogenously during rearrangement of immunoglobulins and T-cell receptor loci (see Chap. 21, Sec. 21.3.1). These animals also show chromosomal instability in their normal cells and are susceptible to lymphoma, suggesting that these act as tumor-suppressor genes (Khanna and Jackson, 2001). Consistent with phenotypes observed in the animals, fibroblasts derived from DNA-PKcs, KU70 or KU80 deficient mice present delayed kinetics of DSB repair and overall lower DSB rejoining following ionizing radiation (see Chap. 15).
The RAD50–MRE11–NBS1 protein complex (termed MRN) acts in both HR and NHEJ pathways (see Figs. 5–7 and 5–8) and also in maintenance of telomeres (see Sec. 5.6). Mutations in the NBS1 gene (also called the p95 or nibrin gene in humans) result in Nijmegen breakage syndrome (NBS), a recessive disorder with some phenotypic similarities to ataxia telangiectasia (AT); (see Sec. 5.5), including chromosomal instability, radiosensitivity, and an increased incidence of lymphoid tumors (Featherstone and Jackson, 1998; Little, 1994). Mutations in human MRE11 have been linked to the ataxia-telangiectasia–like disorder (ATLD). Cells from NBS, AT, and ATLD patients are hypersensitive to DSB-inducing agents and show radioresistant DNA synthesis (persistent DNA synthesis after irradiation that is not observed in normal cells) after exposure to ionizing radiation (Girard et al, 2000). Disruption of the mammalian RAD50 or MRE11 genes results in nonviable mice attesting to their importance in development. Biochemical studies of the yeast and human protein complexes have shown that MRE11 has a 3′ to 5′ Mn2+-dependent exonuclease activity on DNA substrates with blunt or 5′ protruding ends and endonuclease activity on hairpin and single-stranded DNA substrates. This suggests that MRE11 may expose single-stranded regions on DNA DSB. This may promote the use of HR or may activate separate pathways related to NHEJ, called microhomology mediated end joining (MMEJ) and single-strand annealing (SSA) (see Fig. 5–8).
Although DNA DSB repair defects and increased radiosensitivity have been reported for a DNA-PKcs–deficient human glioblastoma tumor cell line, there are no human syndromes attributed to defects in DNA-PK protein function. The relative levels of DNA-PKcs protein are generally lower in rodent than in human tissues, and DNA-PKcs and KU80 protein expression varies widely among different tissue types. Evidence suggests that there is no simple relationship between tumor cell radiosensitivity and the absolute level of ATM or DNA-PK protein expression (Chan et al, 1998).
Unlike HR, NHEJ does not require homology and the NHEJ proteins simply link the ends of DNA breaks together; this usually results in the loss or gain of a few nucleotides during modification of the damaged DNA to produce ligatable ends (5′-phosphate and 3′-hydroxyl). NHEJ is therefore an error-prone pathway, but is operational throughout the cell cycle. There is evidence that RAD52 (a HR-related protein), and the KU70/80 heterodimer, a DNA end-binding protein that functions in NHEJ, compete for binding to DSBs and channel the repair of DSBs into HR or NHEJ respectively, depending on the cellular context (van Gent et al, 2001). The BRCA1 and 53BP1 proteins are also suggested to direct the choice of DNA repair pathway (Bouwman et al, 2010).
5.3.6 DNA Crosslink Repair: Fanconi Anemia Proteins
The Fanconi anemia pathway is a specialized pathway for the repair of interstrand crosslinks that can occur during S-phase. The importance of this pathway is highlighted by patients with predisposition to cancer and deficiencies in 1 of 13 Fanconi anemia complementation (FANC) proteins. In this pathway, unique FANC proteins coordinate a repair mechanism including components of translesion synthesis, HR and NER (Fig. 5–9). When a replication fork approaches an interstrand crosslink, the DNA is not able to form an open configuration for the passage of the polymerase. The FANCM protein initially recognizes the lesion, which recruits an FA-core complex and creates a large E3 ubiquitin ligase that catalyses the ubiquitylation of FANCD2 and FANCI proteins that subsequently localize to the damage site and coordinate downstream functions of nucleases (eg, FAN1), DNA polymerases, NER components, and BRCA1/2-mediated HR (Kee and D'Andrea, 2010). Together these mechanisms lead to the restart of the replication fork, thereby preventing cell death or genomic rearrangements (Moldovan and D'Andrea, 2009).
Fanconi anemia pathway for repairing interstrand crosslinks. Large protein complexes mediate recognition and repair of interstrand crosslinks by the Fanconi anemia pathway. Initially, when replicating DNA meets an interstrand crosslink, the FANCM protein complex recognizes the lesion and recruits the "FA core complex" consisting of FANCs A, B, C, L, E, F, and G. This induces ubiquitylation of FANCD2 and FANCI, facilitating the downstream processes. The crosslink is then unhooked by FAN1, Mus81/Eme1, and ERCC1/XPF1. Although the exact mechanism is poorly understood, Rev1 and Polζ replicate across the lesion. The NER pathway removes the "flipped out" base and repairs the lesion while HR pathway components restore the replication fork after the DSB is resolved. (Adapted from Kee and D'Andrea, 2010.)