Achromosomal double-strand break (DSB) can arise from multiple sources including ionizing radiation and DNA replication itself. An understanding of the intricate pro tein pathways that recognize DSBs and recruit the DNA repair and cell cycle checkpoint machinery is developing rapidly. The ATM kinase plays an early, pivotal role in the signaling process by detecting DSBs and relaying this information to numerous downstream transducer and effector proteins. Within minutes after DSBs occur, ATM undergoes inter-molecular autophosphorylation at Ser¹⁹⁸¹, which converts it to an active monomer. ATM^Ser1981-P immediately phosphorylates histone H2AX over a megabase region of DNA surrounding a DSB. Discrete nuclear foci of phosphorylated H2AX (γH2AX) are visible by immunofluorescence and appear to be true markers of DSBs. MDC1 and 53BP1, transducer proteins that contain two C-terminal BRCT domains, are also phosphorylated by ATM and colocalize faithfully with γH2AX. Subsequent transducers and effectors include the Mre11-Rad50-NBS1 complex (both transducer and effector), and the breast cancer susceptibility proteins BRCA1 (a transducer) and BRCA2 (an effector). BRCA2 interacts directly with DNA and the Rad51 strand-transferase to help initiate homologous recombination. When the DNA replication machinery is chemically inhibited or encounters a damaged template containing single-strand breaks or blocking lesions, replication forks may arrest, collapse into one-sided DSBs, and require recombinational repair to be reestablished. This recovery process is dependent on the ATR kinase acting in concert with the Rad17-Rfc clamp-loader complex and the Rad9-Rad1-Hus1 clamp complex. Modifiers of DNA topology, such as BLM and WRN helicases associated with Bloom and Werner syndromes, assist in preserving chromosomal continuity during replication. These proteins are thought to resolve anomalous replication intermediates that arise at stalled forks, thereby preventing aberrant recombination for unrepaired DSBs. Overall, the precise nature of a DSB likely determines whether ATM or ATR is utilized to initiate the damage-response pathways.

Replication-Independent Double-Strand Breaks (DSBs)

DNA Repair Systems that Act on DSBs

Cells possess complex, highly efficient mechanisms for detecting DSBs and signaling their presence to the DNA repair and replication machinery. In this review, we address what is known about these recognition and information transfer systems outlined in (Fig. 1). Our understanding of how cells respond to DSBs has developed rapidly with respect to the enzymatic machinery that performs repair. Considerably less is known about the preceding events of detecting/sensing/recognizing breaks and the signaling processes that recruit DNA repair systems to the sites of damage. The two major pathways that repair DSBs are referred to as nonhomologous end joining (NHEJ)^21,22 and homologous recombinational repair (HRR).²³²⁵ In this review, we address what is known about these recognition and information transfer systems, along with a brief, updated summary of HRR (Fig. 5).

Figure 1

Recognition and signaling of DSBs. DSBs can arise directly through the action of radiation or chemicals, or indirectly though the enzymatic action of DNA repair enzymes on clustered oxidative lesions or the interaction of DNA replication forks with single-strand (more...)

Figure 5

Outline of events in HRR. BRCA1 has an important, undefined role in regulating HRR. The MRN complex likely participates in both the recognition and processing of DNA ends to produce single-stranded tails, which will bind avidly to RPA. Formation of the (more...)

DSBs occur normally during meiosis to initiate strand exchange between homologous chromosomes and in hematopoietic cells during gene processing through V(D)J recombination, which mediates antibody diversity and gene rearrangements for T cell receptors. These highly regulated, programmed DSB-mediated processes are normally extremely accurate and utilize many of the same DNA break-processing enzymes that repair spontaneous or agent-induced DSBs. High levels of spontaneous DSBs and chromosomal rearrangements are observed in mouse cells carrying null mutations in the genes of the NHEJ complexes composed of Ku70-Ku86-DNA-PKcs or LIG4-XRCC4.²⁶²⁸ The levels of chromosomal breaks can be reduced by lowering the oxygen tension from 20% to 3%.²⁹ Elevating the level of reactive oxidative species, rather surprisingly by overexpressing a transgene for the antioxidant enzyme superoxide dismutase 1 (SOD1), increases chromosome breakage. In SOD1-overexpressing cells, reducing oxygen to 3% also reduced chromosomal aberrations. The observation that oxidative damage results in spontaneous chromosome breaks may explain the neuronal degeneration and premature aging that typify mice having NHEJ mutations.³⁰³⁴

However, not all NHEJ-defective cell lines (i.e., Ku70 and Ku80 mutants in hamster CHO and chicken DT40 backgrounds) display markedly increased levels of spontaneous chromosomal aberrations.³⁵³⁷ The reason for the significant differences among cell types is not clear, but it is noteworthy that both the CHO and DT40 lines are defective for Tp53. Perhaps this defect allows for increased DNA-PKcs-independent end joining in the absence of the DNA-PK or LIG4-XRCC4 complexes.

Central Role of the ATM Kinase in DSB Signaling

The large ATM (ataxia telangiectasia mutated) and ATR (AT and Rad3-related) kinases have come into focus as early, central participants in the DNA damage recognition and signaling processes (Fig. 2 and Fig. 6).^3,3841These functionally related proteins phosphorylate a multitude of substrates and appear to exist in vivo in high molecular weight complexes of >2 × 10⁶ Da,^42,43 which may contain many other damage-response proteins.⁴⁴ Figure 2 expands the theme of (Fig. 1) by depicting numerous phosphorylation events as well as functionally important protein interactions. Exposure of cells to IR immediately activates the ATM kinase (3056 a.a.; Tel1^Sp and Tel1^Sc homologs in yeasts),^45,46 and ATP can also induce activation of by a mechanism involving autophosphorylation.⁴⁷ A major advance came with the discovery that IR-induced activation occurs through intermolecular autophosphorylation of Ser¹⁹⁸¹, which causes dissociation of ATM dimers and enhancement of kinase activity.⁴⁸ After IR, phosphorylation of Ser¹⁹⁸¹ is maximal within 5 min and saturates at a dose of ˜40 cGy.⁴⁸ Upon activation, ATM phosphorylates histone H2AX (modification referred to as γH2AX);⁴⁹ DNA-PK and ATR also contribute to this modification.⁵⁰⁵³ Although ATM binds preferentially to DNA ends in vitro,⁵⁴ the in vivo activation likely results from changes in chromatin structure instead of DNA binding.⁴⁸ ATM phosphorylates numerous key proteins that often appear in nuclear foci (described below) and that mediate checkpoints and DNA repair: namely 53BP1, MDC1/NFBD1, Chk1, Chk2, NBS1, BRCA1, and FANCD2. Altogether, ATM has more than 20 substrates, as recently reviewed in more detail.²⁵ Thus, throughout the cell cycle ATM acts as a master regulator and coordinator in the initial response to DSBs that are not associated with replication forks. ATM is also activated by agents such as methylating chemicals that do not directly cause DSBs, but lead to lesions that are subsequently converted to DSBs.⁵⁵ The closely related ATR kinase discussed below may serve as a partial backup system for ATM and help to reinforce at later times the phosphorylating signaling initiated by ATM (see discussion in ref.41).

Figure 2

ATM mediated phosphorylation events and interactions that implement signaling, repair, and checkpoint functions in response to replication-independent DSBs. Within 60 sec after irradiation, phosphorylation of histone H2AX (referred to as γH2AX) (more...)

Figure 6

ATR-mediated phosphorylation events and interactions that implement signaling, repair, and checkpoint functions in response to replication-associated DSBs. ATR has diverse substrates that overlap with those of ATM. Proteins with names in red are involved (more...)

The fact that ATM is responsible for phosphorylating proteins that implement repair and checkpoint functions suggests that ATM itself might concentrate at sites of DSBs. Indeed, within 5 min after IR, the Ser¹⁹⁸¹-phosphorylated form of ATM begins to form foci that colocalize with γH2AX foci, and these become distinct foci by 60 min.⁴⁸ Under conditions of detergent extraction to remove nucleoplasmic proteins, a portion of the total ATM pool becomes resistant to extraction and is detected in nuclear aggregates immediately after DSB formation.⁵⁶ These aggregates are much more diffuse than the distinct foci formed by γH2AX.

Although the sensor proteins that first recognize DSBs are not well understood, ATM is a major candidate sensor protein. It could act alone or in combination with other proteins discussed below such as 53BP1⁵⁷ and MDC1/NFBD1⁵⁸ that localize within minutes to sites of DSBs. It is noteworthy that certain ATM mutations display a dominant negative phenotype in the heterozygous state, both in humans and mice.^59,60 This situation could arise if mutant ATM binds and sequesters partner proteins into dysfunctional complexes that compete with normal complexes for DNA substrates. Although ATM is required to phosphorylate both NBS1 and Mre11,⁶¹⁶⁵ genetic evidence suggests that the Mre11-Rad50-NBS1 (MRN) complex acts upstream of ATM, at least for some signaling events.⁶⁶ Mre11-defective human cells show reductions in detergent-resistant retention of ATM protein, ATM kinase activity, and phosphorylation of downstream targets.

Origins of Nuclear Foci that Form in Response to DSBs

When cells are exposed to DNA damaging agents, the redistribution and subnuclear localization of specific proteins can be monitored to infer which proteins are important in damage recognition, signaling, checkpoint implementation, and repair. In mammalian cells, Rad51 protein, involved in homologous recombination, was one of the first proteins detected in discrete nuclear foci using immunofluorescence on mitotic and meiotic cells^67,68 (Fig. 3A). The cytological visibility of these foci can be readily explained by the fact that Rad51 forms nucleoprotein filaments that can contain hundreds of Rad51 molecules. It is estimated that ˜100 fluorophore molecules localized within a very small volume are necessary for a visible focus.⁶⁹ Many other proteins discussed below also form foci, but not all of these are expected to assemble en masse as multimeric functional complexes like Rad51. For example, the MRN complex is a key component in the processing of DSB termini,⁷⁰⁷² and the MRN complex forms foci. Although the precise biochemical roles of this complex are not understood, only one or a few of these complexes, as a catalytic component, may be needed to produce single-stranded tails at the termini of DSBs prior to their repair by homologous recombination. Yet, focus formation may arise from the creation of multiple high-affinity binding sites for MRN in the vicinity of the DSB, thus causing numerous MRN complexes to concentrate at the modified site. This idea is illustrated by the observation that foci of Rad52 and Rad54 are highly dynamic structures.⁷³ These foci exhibit rapid exchange of these proteins, which are recruited independently with differing mobility.

Figure 3

Rad51 and γH2AX nuclear foci produced by ionizing radiation. (A) CHO AA8 cells irradiated with 10 Gy and stained with Rad51 antibody and DAPI after 4 h. (B) Unirradiated CHO AA8 cells stained with γH2AX antibody. Putative S phase cells (more...)

Recruitment and CoLocalization of Signaling and Repair Proteins to Sites of γH2AX Foci

In this section we outline the characteristics of many proteins that are implicated in signaling, checkpoints, and repair through their redistribution within the nucleus in response to DNA breakage. Some of these proteins, such as MDC1/NFBD1 and 53BP1, appear to arise as quickly as, and coincident with, γH2AX foci while other foci (e.g., Rad51) arise much later. By further example, colocalizing γH2AX-BRCA1 foci were reported to appear sooner than γH2AX-Rad50 foci,⁷⁵ implying that BRCA1 may act upstream of the Rad50 complex (MRN, discussed below). Figure 4 summarizes available information on the order of appearance of foci, including colocalizations. Certainly there are numerous pitfalls in deciphering the significance of focus formation. Foci studies performed at high doses (e.g., > 5 Gy) and many hours after exposure will be much more difficult to interpret than studies terminated minutes after irradiation at low doses (e.g., < 1 Gy). The remainder of this section summarizes the characteristics and significance of many proteins that have been shown to form nuclear foci.

Figure 4

Timeline of recruitment of signaling, repair, and checkpoint proteins to sites of DSBs. Immediately after irradiation the formation of phosphorylated histone H2AX (γH2AX) is detectable and appears to be a/the critical event that modifies chromatin (more...)

MDC1/NFBD1

Very recently a new nuclear human protein, MDC1 (mediator of DNA checkpoint; also called NFBD1 for nuclear factor containing two BRCT domains at the C-terminus; 2089 a.a.), which constitutively binds to chromatin, was identified as a very early participant in the recognition and signaling process.^58,7982 Because of its C-terminal BRCT domains, MDC1 is a candidate functional homolog of Rad9^Sc, one of the first checkpoint proteins to be identified in budding yeast.^83,84 After IR damage, MDC1 becomes hyperphosphorylated in an ATM-, NBS1-, and Chk2-dependent manner,^58,80,81 but MDC1 focus formation is still seen in the absence of ATM.⁸¹ Within 1 min after irradiation, MDC1 forms visible foci that peak in frequency at 30 min.^58,82 MDC1 foci colocalize precisely with γH2AX foci, and γH2AX is needed for MDC1 focus formation.^58,82 H2AX and MDC1 are mutually interdependent for phosphorylation and focus formation, and MDC1 forms complexes with γH2AX.⁵⁸ However, there are conflicting data concerning whether MDC1 is required for 53BP1 focus formation.^58,82 Suppression of MDC1 by siRNA results in decreased phosphorylation of SMC1^S996 and Chk1^S345.⁵⁸ This defect in Chk1 phosphorylation results in defective intra-S phase and G2 checkpoints after IR exposure. Suppression of MDC1 expression by siRNA also causes reduced apoptosis in response to IR damage (because of the loss of the MDC1-Chk2^P68 interaction⁸⁰), but also decreases colony-forming ability.⁸²

53BP1

Human 53BP1 (1972 a.a. and containing two C-terminal BRCT⁸⁵ repeats like MDC1/NFBD1) was identified as a Tp53-interacting protein in a yeast two-hybrid screen^86,87 and was found to have homology with Rad9^Sc.⁸⁸ 53BP1-null (or 53BP1-truncated) mice have a phenotype resembling that of H2AX-deficient mice and that of AT, i.e., growth retardation, immune deficiency, radiation sensitivity, impaired Chk2 phosphorylation/activation (Fig. 2), and cancer proneness.^89,90 Although embryo-derived cultures appear to have intact G1, S, and G2 checkpoints,⁸⁹ other studies with different cell types report a requirement for 53BP1 in the S and G2 checkpoints.^57,78

Cytologically, 53BP1 shows diffuse nuclear immunostaining in undamaged G1 cells but a punctate pattern in S phase,⁹⁰ suggesting localization to sites of stalled or broken replication forks. 53BP1 localizes into nuclear foci in most cells as early as five min after IR exposure; doses as low as 0.5 Gy result in formation of these foci.⁸⁸ After 1 Gy, the maximal numbers of foci per cell (≇ 20–35) and of foci-positive cells (˜92%) are seen between 15 and 120 min.^78,88,91 This number of foci is consistent with the expected yield of DSBs, as discussed above. Significant colocalization of 53BP1 with γH2AX is seen between 10 and 240 min,^78,88,91 and the two proteins show damage-dependent coimmunoprecipitation.⁹¹ As with γH2AX, 53BP1 focus formation is specific for agents that produce DSBs and occurs more rapidly than that of other proteins discussed below (BRCA1, MRN, and Rad51). Phosphorylation and focus formation of 53BP1 are controlled independently.⁹² One notable difference between 53BP1 and γH2AX is that only γH2AX forms foci in mitosis,^50,93 where 53BP1 associates with kinetocores.⁹⁴

Recent work shows that 53BP1 has a central role in IR-induced DSB signaling for the Sand G2-phase checkpoints [see editorial in ref. 95]. Upon inhibition of 53BP1 by siRNA, phosphorylation of Tp53 and BRCA1 by ATM is blocked, and the downstream formation of IR-induced BRCA1 foci is largely abolished.⁵⁷ Studies with mouse knockout cells show that 53BP1 is required for a normal G2-M checkpoint at low IR doses.⁷⁸ In AT cells, 53BP1 focus formation after 1 Gy was diminished at early times (10–20 min post irradiation) in one study,⁹¹ and the hyperphosphorylation that occurs after irradiation was absent or reduced.^91,93 However, in other studies that used 3 or 8 Gy, 53BP1 focus formation appeared to be normal in AT cells,^88,96 perhaps because of the higher doses used. ATM phosphorylates 53BP1 in vitro,^78,91,92 further suggesting that ATM is responsible for directly phosphorylating 53BP1 within γH2AX foci. ATM and 53BP1 also show IR-dependent coimmunoprecipitation.⁹⁶ 53BP1 may either help recruit and activate ATM at sites of DSBs, or recruit ATM substrates to these sites.⁹⁶ It is noteworthy that 53BP1, as well as Chk2, appears to be present in γH2AX foci before any of the proteins that participate in DSB repair. These results suggest that the initiation of checkpoints precedes the onset of DSB repair, which occurs over a period of several hours, depending on the dose.

HDAC4

Histone deacetylase 4 (HDAC4) is another early participant in IR-induced focus formation, and HDAC4 foci are seen in cells defective in ATM, DNA-PKcs, or NBS1.⁹⁷ In contrast, HDAC2 or HDAC6 do not form foci. Interestingly, the stability of 53BP1, as well as its focus formation, shows a dependence on the presence of HDAC4, as shown by siRNA inhibition experiments. Depletion of HDAC4 abrogates the G2 delay and confers sensitization to killing by IR while also reducing cell viability.⁹⁷ In this study the authors suggested that the degree of persistence of HDAC4 foci might be a measure of cellular radiosensitivity.

ChK2

Chk2/Cds1 is a key checkpoint kinase (for reviews see refs. 3,98,99), whose role appears to be primarily promoting apoptosis, not cell survival, after IR exposure.^100,101 Chk2-deficient mice are radioresistant and defective in Tp53-mediated transcriptional changes.^100,101 Thymocytes, splenocytes, skin, and neurons in the developing brain show protection from IR-induced apoptosis. The G1 checkpoint, but not the G2 or S-phase checkpoints, was substantially impaired in Chk2^-/- embryonic fibroblasts and ES cells.^100,101 IR-induced stabilization of Tp53 in Chk2^-/- cells is ˜60% of that in wild-type cells.¹⁰¹ Caffeine further reduces Tp53 accumulation, suggesting the presence of another pathway for Tp53 stabilization that is ATM/ATR-dependent, but Chk2-independent. In spite of Tp53's partial stabilization and phosphorylation at Ser²³ (Ser²⁰ in human cells) in the absence of Chk2, Tp53-dependent transcriptional induction of target genes, such as CDKN1A/p21, was not observed in Chk2^-/- cells.

IR treatment results in ATM-dependent activation and phosphorylation of Chk2 at Thr⁶⁸.¹⁰² The complete activation of Chk2 requires NBS1.^103,104 Immuno-staining using phospho-specific Chk2 antibodies suggests that Chk2-Thr^68P localizes into discrete foci within 10 min after irradiation and colocalizes with both γH2AX and 53BP1 (Fig. 4).^57,102 These Chk2-Thr68P foci did not appear in AT cells or in 53BP1-depleted cells, and nonphosphorylated Chk2 molecules remain distributed throughout the nucleus.⁵⁷ However, a subsequent study using 53BP1 mouse knockout cells and a different Chk2-Thr^68P antibody concluded that 53BP1 is not required for Chk2 activation.⁷⁸ These different conclusions might be accounted for by inadequate specificity of the Chk2-Thr^68P antibody¹⁰² used in the former studies (see commentary in ref. 95). In summary, phosphorylation and focus formation of Chk2 may be a major determinant in programming cells for elimination by apoptosis through Chk2's site-specific phosphorylation of Tp53.

Chk1

Few Chk1 foci studies have been reported, but Chk1 and BRCA1 foci colocalize in the absence of IR exposure.¹⁰⁵ In comparison to Chk2 described above, after IR damage in chicken DT40 cells Chk1 promotes reproductive survival, apoptosis (in these Tp53-deficient cells), implementation of the G2 checkpoint, and phosphorylation of Cdc2.¹⁰⁶ Notably, Chk1 null cells completely lose the G2 checkpoint in DT40 cells (see Fig. 2). Inhibition of human Chk1 with siRNA also results in a G2 checkpoint defect, and phosphorylation of Ser³¹⁷ and Ser³⁴⁵ appears nonessential for IR-mediated activation of Chk1 and the G2 checkpoint.¹⁰⁷ IR activation of Chk1 depends on BRCA1.¹⁰⁵

MRN Complex

The analysis of nuclear focus formation by various damage-response proteins suggests that γH2AX formation plays a critical role in recruiting and assembling repair proteins, including the Mre11-Rad50-NBS1 (MRN) complex. This complex is essential for HRR in human cells since hypomorphic mutations confer radiosensitivity.¹⁰⁸¹¹⁰ MRN localizes to sites of damage within 30 min after irradiation¹¹¹ (Fig. 4), and recent genetic evidence suggests that it may be a primary DSB recognition factor.⁶⁶ By using a 390-nm laser combined with BrdUrd incorporation and Hoechst dye 33258¹¹² to produce striped regions of DSBs, Bonner and coworkers have shown in human breast tumor MCF7 cells that γH2AX stripes appear rapidly in all cells, and MRN colocalizes to these stripes within 30 min.⁷⁵ Colocalization of NBS1 and γH2AX foci appears to involve a direct interaction between NBS1 and γH2AX (and not H2AX), which is mediated by the FHA/BRCT domain of NBS1.⁵²

Initial observations of MRN foci indicated that the kinetics of appearance was too delayed to correspond with productive repair of DSBs.¹¹³ However, the patterns of focus formation of proteins such as Rad50 and Rad51 are strongly influenced by the method of preparing the cells. In earlier experiments using methanol fixation and acetone to permeabilize human diploid fibroblasts, the percentage of nuclei that were positive for Rad50 focus formation after 12 Gy γ-irradiation reached a maximum of ˜65% after 8 hr.¹¹³ In comparison, nuclei with Rad51 foci reached a broader maximum between 4–8 hr at ˜35%. Subsequent studies in detergent extracted cells, designed to reveal proteins tightly associated with chromatin, found that MRN foci were detectable within 10 min after irradiation and reached a maximum at 2 hr when 70–95% of cells were positive.¹¹⁴ Since MRN foci are present in AT cells, MRN focus formation does not depend on the phosphorylation of NBS1 by ATM.⁶¹⁶⁴ These results suggest that the MRN complex may participate in a very early, ATM-independent step of DSB recognition.⁶⁶ Larger aggregates of MRN foci that become apparent by 8 hr in normal cells were not seen in AT cells.¹¹⁴ These larger, more robust foci may represent sites of slow or abortive repair of complex DSBs. In unirradiated detergent-extracted cells, Mre11 shows a high degree of colocalization with immunostaining of PML bodies, a nuclear depot of many proteins that may help regulate cellular defense against insults such as viruses.¹¹⁵

BRCA1 and Rad51

Radiation-induced γH2AX focus formation occurs within several minutes in both MCF7 and IMR90 human cells after exposure to only 0.6 Gy.⁵⁰ However, higher radiation doses are usually needed to visualize the foci formed by several key proteins that are recruited to the γH2AX foci. After 12 Gy of IR, the kinetics of BRCA1 focus formation is significantly more rapid than that of MRN focus formation.⁷⁵ By 2 hr, 10–15% of IMR90 cells show BRCA1 foci, and these overlap extensively with γH2AX foci.⁵⁰ While γH2AX-BRCA1 colocalization is maximal by 2 hr, γH2AX-MRN colocalization increases up to 8 hr. BRCA1 focus formation and hyperphosphorylation after IR is dependent on MDC1.¹¹⁶ In the absence of detergent extraction, Rad51 foci appear at about the same time as γH2AX-MRN foci, but in different cells.¹¹³ Since MRN acts upstream of Rad51 (see Fig. 5), this observation is paradoxical unless all DSB repair events in a given cell were to occur in a synchronous manner. Rad51 foci become prominent at 6 hr in 20–25% of the cells, when the majority of these foci colocalize with BRCA1 foci. Rad51 foci form relatively slowly, are much less numerous than the estimated numbers of DSBs, and require high doses for their detection. Therefore, their biological significance is still unclear.

In SV40-transformed fibroblasts, the kinetics of focus formation was faster (e.g., γH2AX-BRCA1 colocalization in 45 min) and more cells had Rad51 foci, but the order of foci appearance was the same as for IMR90 cells.⁷⁵ The relatively early appearance of γH2AX-BRCA1 foci suggests that BRCA1 might interact directly with DNA breaks.¹¹⁷

Pretreatment of MCF7 breast carcinoma cells for 30 min with phosphatidylinositol-3-kinase inhibitor wortmannin, which inhibits ATM and DNA-PKcs, completely blocks γH2AX focus formation after irradiation and prevents the formation of repair-protein foci.⁷⁵ Importantly, when added 5 min after irradiation wortmannin has no effect on γH2AX, BRCA1, and Rad51 focus formation. These results emphasize that γH2AX is an early, critical event that initiates DNA repair processes. Consistent with this idea is the finding in mouse H2AX/ knockout B cells or ES cells that BRCA1 and MRN foci cannot form in response to irradiation.^76,77 However, Rad51 foci do form in both these mutant cell types although their intensity seems to be diminished in the mutant ES cells exposed to 20 Gy.^76,77

DNA-PK

The kinase activity of DNA-PK is required for the efficient repair of DSBs by NHEJ, but the molecular mechanism underlying this activity is not understood.^118,119 Very recently the catalytic subunit of DNA-PK (DNA-PKcs) was found to undergo autophosphorylation in vitro at the highly conserved Thr²⁶⁰⁹ and phosphorylation at this position occurred in vivo in response to IR damage.¹²⁰ DNA-PKcs ^Thr2609 colocalizes within 30 min after IR with both γH2AX and 53BP1. After 10 Gy the phosphorylation of Thr²⁶⁰⁹ is maximal by 30 min and persists for up to 4 hr. The biological significance of this phosphorylation is indicated by the finding that a Thr²⁶⁰⁹Ala substitution mutation is associated with defective DSB rejoining and causes increased radiation sensitivity in CHO cells. DNA-PK autophosphorylation at Thr2609is reduced in the absence of ATM. Attempts to identify Ku70/86 foci in irradiated cells have generally been unsuccessful in seeing localization of this DNA end-binding complex.^114,121 Immunostaining of Ku70/86, or total DNA-PKcs, in the absence of detergent extraction, does not reveal discrete foci. This specificity suggests that only DNA-PKcs^Thr2609 may be needed to recruit Ku70/86 to DSBs, and that a high local concentration of Ku70/86 at a break may be unnecessary because of its abundance in the nucleus.

RPA

The trimeric RPA complex binds single-stranded DNA and is an essential component of both HRR and DNA replication. RPA might provide an appropriate marker for foci in which recombination has been initiated through the processing of DSB ends into structures containing single-stranded DNA coated with RPA. In human cells, interaction between RPA and Rad51 is mediated by the 70-kDa subunit of RPA.¹²² RPA foci induced by irradiation are detectable at doses as low as 0.5 Gy and are present at 2 hr (and possibly earlier).¹²³ In focus-positive cells, the number of RPA foci per nucleus reached a maximum at ˜3 hr. It would be of interest to examine the colocalization of RPA with Rad51 at low doses that allow relatively high cell survival (e.g., 0.5 Gy). This information might allow an estimate of the time required for Rad51 filament formation to occur.

TOPBP1

Human TopBP1 (1522 a.a.) has sequence similarity with other checkpoint proteins (Rad4/ Cut5^Sc, Dpb11^Sp, & Mus101^Dm) and contains eight BRCT domains.¹²⁴ TopBP1 binds to DNA ends through its BRCT domains,¹²⁵ is required for DNA replication, and interacts with Polϵ.¹²⁶ After radiation damage, ATM phosphorylates TopBP1, but TopBP1 focus formation occurs even in AT cells.¹²⁷ TopBP1 colocalizes ˜50% with 53BP1 within one hr after irradiation and substantially colocalizes with NBS1 and BRCA1 at six hr after irradiation (Fig. 4). Inhibition of DNA synthesis by hydroxyurea results in relocalization of TopBP1 together with BRCA1 to replication forks, suggesting a role for TopBP1 in rescue of stalled forks.¹²⁶

Replication Associated DSBs (One-Sided Breaks)

Replication-Associated Dsbs Arising from a Damaged Template

In the simplest case, a DSB may occur in one daughter chromatid when a replication fork encounters a SSB, which can arise as an intermediate in base excision repair. Under conditions where excess SSBs are present, as during the repair of methylation damage (e.g., MMS exposure), the production of replication-associated DSBs will be exacerbated. When helicase and polymerase activities become uncoupled at the replication fork¹⁷⁰¹⁷² and generate extended regions of single-stranded DNA (e.g., 1 kb), the likelihood of disrupting chromatid continuity may increase because of increased exposure of SSBs. Moreover, new SSBs might arise from the nicking activity of nucleases acting on single-stranded DNA.

When replication forks encounter polymerase-blocking lesions such as bulky adducts, DSBs can arise by several processes. A blocking lesion in the leading strand can result in the generation of extensive downstream ssDNA in that strand (Fig. 7A). These single-stranded gaps have been documented experimentally during SV40 replication that initiates upstream of a pyrimidine-dimer^173,174 and are favored under conditions when damage bypass of specific lesions is inhibited.^144,175 Under these conditions, ssDNA is generated as the replication fork proceeds while chain extension on the leading strand is restricted. The resultant DNA structures can become destabilized and cause the marked increase in DSBs seen after UV irradiation of both yeast¹⁷⁶ and hamster cells¹⁷⁷ containing photolytic lesions induced by UVA in BrdUrd-substituted DNA in the presence of a photosensitizing dye. This phenomenon is greatly exaggerated in bypass-deficient xeroderma pigmentosum variant (XP-V) cells, which are defective in the bypass polymerase Y/η (discussed further below).^178,179 Regions of ssDNA are also candidates for the formation of secondary structures such as hairpins and cruciform structures that can be recognized and cleaved by the MRN complex or other enzymes.¹⁸⁰¹⁸³

Figure 7

Formation and repair of DSB arising when replication forks encounter damage. (A) Arrest of the replication fork. (B) Fork collapse and DSB generation upon the encounter of the replication fork with a preexisting nick or gap. (C) Formation of a Holliday (more...)

Under certain circumstances stalled replication forks can regress or reverse to generate a 4-stranded structure (often called a “chickenfoot”¹⁸⁴) that can be considered a topologically masked DSB (Fig. 7E). Recent electron micrographs reveal the presence of such structures in yeast rad53 kinase checkpoint mutants.¹⁸⁵ However, in mammalian cells early studies cast doubt on the existence of these structures. The appearance of doubly-dense DNA from cells pulse labeled with BrdUrd was found to decrease dramatically upon crosslinking with psoralen, suggesting that branch migration occurred as an artifact of DNA isolation.^186,187 Regressed forks are structurally similar to, but topologically distinct from, Holliday junctions (HJs), a common intermediate produced during the repair of DSBs by HR (Fig. 7C). These 4-stranded structures generated by fork regression can be unwound by the structure specific helicases WRN and BLM¹⁸⁸¹⁹⁰ and cut by resolvases.^191,192 Thus, branched intermediate structures that arise at sites of stalled replication can be converted to DSBs in a manner dependent upon their structural and topological context, as further addressed below.

Acknowledgments

We thank Karlene Cimprich, John Hinz, David Schild, Robert Tebbs, Alice Yamada, and Dawn Yean for providing valuable comments on the manuscript, and also Joel Bedford, Bill Dewey, and John Ward for helpful discussion. This work was prepared under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and funded by the Low Dose Radiation Research Program, Biological and Environmental Research (BER), U.S. Department of Energy, by NIH/NCI grant P01 CA92584-02, and by ACS grant # RPG-00-036-01 CNE (CLL).

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