Corruption of DNA end-joining in mammalian chromosomes by progerin expression.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." Progerin is also produced in small amounts in healthy individuals by alternative splicing of RNA and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting alteration of DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by nonhomologous end-joining (NHEJ), a non-templated rejoining of DNA ends that can be error-prone; however a good portion of NHEJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin correlated with increased NHEJ relative to HR. We now report on progerin's impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. Two closely spaced DSBs were induced in the integrated substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. DNA sequencing revealed that progerin expression correlated with a significant shift away from precise end-joining between the two I-SceI sites and toward imprecise end-joining. Additional experiments revealed that progerin did not reduce HR fidelity. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, thereby shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to accelerated and normal aging through compromised genome stability.

Alteration of genetic recombination and double-strand break repair in human cells by progerin expression.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal, dominant genetic condition characterized by many features of accelerated aging. On average, children with HGPS live to about fourteen years of age. The syndrome is commonly caused by a point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The LMNA mutation alters splicing, leading to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at very low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting corruption of DNA repair. In this work, we investigated the influence of progerin expression on DSB repair in the human genome at the nucleotide level. We used a model system that involves a reporter DNA substrate inserted in the genome of cultured human cells. A DSB could be induced within the substrate through exogenous expression of endonuclease I-SceI, and DSB repair events occurring via either homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recoverable. Additionally, spontaneous HR events were recoverable in the absence of artificial DSB induction. We compared DSB repair and spontaneous HR in cells overexpressing progerin versus cells expressing no progerin. We report that overexpression of progerin correlated with an increase in DSB repair via NHEJ relative to HR, as well as an increased fraction of HR events occurring via gene conversion. Progerin also engendered an apparent increase in spontaneous HR events, with a highly significant shift toward gene conversion events, and an increase in DNA amplification events. Such influences of progerin on DNA transactions may impact genome stability and contribute to aging.

Mismatch tolerance during homologous recombination in mammalian cells.

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the "recipient") rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a "donor" tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of "homeology" relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.

High homology is not required at the site of strand invasion during recombinational double-strand break repair in mammalian chromosomes.

We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the "recipient") rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated "donor" tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient. Repair events were recoverable by selection for tk-positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Previous work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In the current study we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest a search for high homology prior to recombination that is not mediated by an invading DNA terminus.

Intrachromosomal recombination between highly diverged DNA sequences is enabled in human cells deficient in Bloom helicase.

Mutation of Bloom helicase (BLM) causes Bloom syndrome (BS), a rare human genetic disorder associated with genome instability, elevation of sister chromatid exchanges, and predisposition to cancer. Deficiency in BLM homologs in Drosophila and yeast brings about significantly increased rates of recombination between imperfectly matched sequences ("homeologous recombination," or HeR). To assess whether BLM deficiency provokes an increase in HeR in human cells, we transfected an HeR substrate into a BLM-null cell line derived from a BS patient. The substrate contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI, as well as a functional tk gene to serve as a potential recombination partner for the tk-neo gene. The two tk sequences on the substrate displayed 19% divergence. A double-strand break was introduced by expression of I-SceI and repair events were recovered by selection for G418-resistant clones. Among 181 events recovered, 30 were accomplished via HeR with the balance accomplished by nonhomologous end-joining. The frequency of HeR events in the BS cells was elevated significantly compared to that seen in normal human fibroblasts or in BS cells complemented for BLM expression. We conclude that BLM deficiency enables HeR in human cells.

Induction of recombination between diverged sequences in a mammalian genome by a double-strand break.

To investigate whether mammalian cells can carry out recombinational double-strand break (DSB) repair between highly diverged sequences, mouse fibroblasts were transfected with DNA substrates that contained a "recipient" thymidine kinase (tk) gene disrupted by the recognition site for endonuclease I-SceI. Substrates also contained a linked "donor" tk gene sequence. Following DSB induction by I-SceI, selection for tk-expressing clones allowed recovery of repair events occurring by nonhomologous end-joining or recombination with the donor sequence. Although recombinational repair was most efficient when donor and recipient shared near-perfect homology, we recovered recombination events between recipient and donor sequences displaying 20 % nucleotide mismatch. Recombination between such imperfectly matched ("homeologous") sequences occurred at a frequency of 1.7 × 10(-7) events per cell and constituted 3 % of the DSB repair events recovered with the pair of homeologous sequences. Additional experiments were done with a substrate containing a donor sequence comprised of a region sharing high homology with the recipient and an adjacent region homeologous to the recipient. Recombinational DSB repair tracts initiating within high homology propagated into homeology in 11 of 112 repair events. These collective results contrasted with our earlier work in which spontaneous recombination (not intentionally induced by a DSB) between homeologous sequences occurred at an undetectable frequency of less than 10(-9) events per cell, and in which events initiating within high homology propagated into adjoining homeology in one of 81 events examined. Our current work suggests that homology requirements for recombination are effectively relaxed in proximity to a DSB in a mammalian genome.

Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics.

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.

Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes.

Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.

Wild-type p53-induced phosphatase 1 dephosphorylates histone variant gamma-H2AX and suppresses DNA double strand break repair.

In response to DNA double strand breaks, the histone variant H2AX at the break site is phosphorylated at serine 139 by DNA damage sensor kinases such as ataxia telangiectasia-mutated, forming gamma-H2AX. This phosphorylation event is critical for sustained recruitment of other proteins to repair the break. After repair, restoration of the cell to a prestress state is associated with gamma-H2AX dephosphorylation and dissolution of gamma-H2AX-associated damage foci. The phosphatases PP2A and PP4 have previously been shown to dephosphorylate gamma-H2AX. Here, we demonstrate that the wild-type p53-induced phosphatase 1 (WIP1) also dephosphorylates gamma-H2AX at serine 139 in vitro and in vivo. Overexpression of WIP1 reduces formation of gamma-H2AX foci in response to ionizing and ultraviolet radiation and blocks recruitment of MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1) to DNA damage foci. Finally, these inhibitory effects of WIP1 on gamma-H2AX are accompanied by WIP1 suppression of DNA double strand break repair. Thus, WIP1 has a homeostatic role in reversing the effects of ataxia telangiectasia-mutated phosphorylation of H2AX.

Ensuring the fidelity of recombination in mammalian chromosomes.

Mammalian cells frequently depend on homologous recombination (HR) to repair DNA damage accurately and to help rescue stalled or collapsed replication forks. The essence of HR is an exchange of nucleotides between identical or nearly identical sequences. Although HR fulfills important biological roles, recombination between inappropriate sequence partners can lead to translocations or other deleterious rearrangements and such events must be avoided. For example, the recombination machinery must follow stringent rules to preclude recombination between the many repetitive elements in a mammalian genome that share significant but imperfect homology. This paper takes a conceptual approach in addressing the homology requirements for recombination in mammalian genomes as well as the general strategy used by cells to reject recombination between similar but imperfectly matched sequences. A mechanism of heteroduplex rejection that involves the unwinding of recombination intermediates that may form between mismatched sequences is discussed.

DNA damage and homologous recombination signaling induced by thymidylate deprivation.

DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC(50) doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, gamma-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.

Accurate homologous recombination is a prominent double-strand break repair pathway in mammalian chromosomes and is modulated by mismatch repair protein Msh2.

We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a "donor" tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.

Genetic exchange between homeologous sequences in mammalian chromosomes is averted by local homology requirements for initiation and resolution of recombination.

We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a "recipient" gene. Each substrate also contained one of several "donor" tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologous sequence sharing only 80% identity with the recipient gene. Mouse Ltk(-) fibroblasts were stably transfected with the various substrates and tk(+) segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

A role for DNA mismatch repair protein Msh2 in error-prone double-strand-break repair in mammalian chromosomes.

We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."

Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1.

We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.

Determination of intrachromosomal recombination rates in cultured mammalian cells.

Recombination is involved in many important biological processes including DNA repair, gene expression, and generation of genetic diversity. Recombination must be carefully regulated so as to prevent the deleterious consequences that may result from rearrangements between dissimilar sequences in a genome. It is of considerable interest to study the mechanisms by which genetic rearrangements in mammalian chromosomes are regulated in order to understand better how genomic integrity is normally maintained and to gain insight into the types of genetic mutations that may destabilize the genome. To explore such issues in mammalian chromosomes, a suitable experimental system must be developed. In this chapter, we describe a model system for studying intrachromosomal recombination in cultured mammalian cells. We discuss two model recombination substrates, a method for stably introducing the substrates into cultured Chinese hamster ovary cells, and a method for determining rates of intrachromosomal recombination between sequences contained within the integrated substrates. The general approach described here should be applicable to the study of a variety of aspects of recombination in virtually any cultured mammalian cell line.

Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha, a chemical inhibitor of p53.

We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.