XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.DNA double-strand breaks (DSBs) are potentially dangerous lesions, since their misrepair may cause chromosomal translocations, gene amplifications, loss of heterozygosity (LOH), and other types of genomic instability characteristic of human cancers (7, 9, 21, 40, 76, 79). DSBs are repaired predominantly by nonhomologous end joining or homologous recombination (HR), two evolutionarily conserved DSB repair mechanisms (8, 12, 16, 33, 48, 60, 71). DSBs generated during the S or G₂ phase of the cell cycle may be repaired preferentially by HR, using the intact sister chromatid as a template for repair (12, 26, 29, 32, 71). Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DSBs, which has led to the proposal that SCR protects against genomic instability, cancer, and aging. Indeed, a number of human cancer predisposition genes are implicated in SCR control (10, 24, 45, 57, 75).HR entails an initial processing of the DSB to generate a free 3′ single-stranded DNA (ssDNA) overhang (25, 48, 56). This is coupled to the loading of Rad51, the eukaryotic homolog of Escherichia coli RecA, which polymerizes to form an ssDNA-Rad51 “presynaptic” nucleoprotein filament. Formation of the presynaptic filament is tightly regulated and requires the concerted action of a large number of gene products (55, 66, 68). Rad51-coated ssDNA engages in a homology search by invading homologous duplex DNA. If sufficient homology exists between the invading and invaded strands, a triple-stranded synapse (D-loop) forms, and the 3′ end of the invading (nascent) strand is extended, using the donor as a template for gene conversion. This recombination intermediate is thought to be channeled into one of the following two major subpathways: classical gap repair or synthesis-dependent strand annealing (SDSA) (48). Gap repair entails the formation of a double Holliday junction, which may resolve into either crossover or noncrossover products. Although this is a major pathway in meiotic recombination, crossing-over is highly suppressed in somatic eukaryotic cells (26, 44, 48). Indeed, the donor DNA molecule is seldom rearranged during somatic HR, suggesting that SDSA is the major pathway for the repair of somatic DSBs (26, 44, 49, 69). SDSA terminates when the nascent strand is displaced from the D-loop and pairs with the second end of the DSB to form a noncrossover product. The mechanisms underlying displacement of the nascent strand are not well understood. However, failure to displace the nascent strand might be expected to result in the production of longer gene conversion tracts during HR (36, 44, 48, 63).Gene conversion triggered in response to a Saccharomyces cerevisiae or mammalian chromosomal DSB generally results in the copying of a short (50- to 300-bp) stretch of information from the donor (short-tract gene conversion [STGC]) (14, 47, 48, 67, 69). A minority of gene conversions in mammalian cells entail more-extensive copying, generating gene conversion tracts that are up to several kilobases in length (long-tract gene conversion [LTGC]) (26, 44, 51, 54, 64). In yeast, very long gene conversions can result from break-induced replication (BIR), a highly processive form of gene conversion in which a bona fide replication fork is thought to be established at the recombination synapse (11, 36, 37, 39, 61, 63). In contrast, SDSA does not require lagging-strand polymerases and appears to be much less processive than a conventional replication fork (37, 42, 78). BIR in yeast has been proposed to play a role in LOH in aging yeast, telomere maintenance, and palindromic gene amplification (5, 41, 52). It is unclear to what extent a BIR-like mechanism operates in mammalian cells, although BIR has been invoked to explain telomere elongation in tumors lacking telomerase (13). It is currently unknown whether LTGC and STGC in somatic mammalian cells are products of mechanistically distinct pathways or whether they represent alternative outcomes of a common SDSA pathway.Vertebrate cells contain five Rad51 paralogs—polypeptides with limited sequence homology to Rad51—Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3 (74). The Rad51 paralogs form the following two major complexes: Rad51B/Rad51C/Rad51D/XRCC2 (BCDX2) and Rad51C/XRCC3 (CX3) (38, 73). Genetic deletion of any one of the rad51 paralogs in the mouse germ line produces early embryonic lethality, and mouse or chicken cells lacking any of the rad51 paralogs reveal hypersensitivity to DNA-damaging agents, reduced frequencies of HR and of sister chromatid exchanges, increased chromatid-type errors, and defective sister chromatid cohesion (18, 72, 73, 82). Collectively, these data implicate the Rad51 paralogs in SCR regulation. The purified Rad51B/Rad51C complex has been shown to assist Rad51-mediated strand exchange (62). XRCC3 null or Rad51C null hamster cells reveal a bias toward production of longer gene conversion tracts, suggesting a role for the CX3 complex in late stages of SDSA (6, 44). Rad51C copurifies with branch migration and Holliday junction resolution activities in mammalian cell extracts (35), and XRCC3, but not XRCC2, facilitates telomere shortening by reciprocal crossing-over in telomeric T loops (77). These data, taken together with the meiotic defects observed in Rad51C hypomorphic mice, suggest a specialized role for CX3, but not for BCDX2, in resolving Holliday junction structures (31, 58).To further address the roles of Rad51 paralogs in late stages of recombination, we have studied the balance between long-tract (>1-kb) and short-tract (<1-kb) SCR in XRCC2 mutant hamster cells. We found that DSB-induced gene conversion in both XRCC2 and XRCC3 mutant cells is biased in favor of LTGC. These defects were suppressed by expression of wild-type (wt) XRCC2 or XRCC3, respectively, although the dependence upon ATP binding and hydrolysis differed between the two Rad51 paralogs. These results indicate that Rad51 paralogs play a more general role in determining the balance between STGC and LTGC than was previously appreciated and suggest roles for both the BCDX2 and CX3 complexes in influencing the termination of gene conversion in mammals.     

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G₁, consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G₂, specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3′ overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G₂ through end resection and induction of NHEJ in G₁ through ATM-dependent signaling.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2^F/− MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to their fusion (18). Telomere NHEJ is also accelerated by the ATM phosphorylation target MDC1, which is required for the prolonged association of 53BP1 at sites of DNA damage (19).Although loss of TRF2 leads to telomere deprotection at all stages of the cell cycle, NHEJ of uncapped telomeres takes place primarily before their replication in G₁ (25). Postreplicative (G₂) telomere fusions can occur at a low frequency upon TRF2 deletion, but only when cyclin-dependent kinase activity is inhibited with roscovitine (25). The target of Cdk1 in this setting is not known.Here, we dissect the role of the MRN (Mre11/Rad50/Nbs1) complex and H2AX at telomeres rendered dysfunctional through deletion of TRF2. The highly conserved MRN complex has been proposed to function as the double-stranded break (DSB) sensor in the ATM pathway (reviewed in references 34 and 35). In support of this model, Mre11 interacts directly with DNA ends via two carboxy-terminal DNA binding domains (13, 14); the recruitment of MRN to sites of damage is independent of ATM signaling, as it occurs in the presence of the phosphoinositide-3-kinase-related protein kinase inhibitor caffeine (29, 44); in vitro analysis has demonstrated that MRN is required for activation of ATM by linear DNAs (27); a mutant form of Rad50 (Rad50S) can induce ATM signaling in the absence of DNA damage (31); and phosphorylation of ATM targets in response to ionizing radiation is completely abrogated upon deletion of NBS1 from MEFs (17). These data and the striking similarities between syndromes caused by mutations in ATM, Nbs1, and Mre11 (ataxia telangiectasia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disease, respectively) are consistent with a sensor function for MRN.MRN has also been implicated in several aspects of DNA repair. Potentially relevant to DNA repair events, Mre11 dimers can bridge and align the two DNA ends in vitro (49) and Rad50 may promote long-range tethering of sister chromatids (24, 50). In addition, a binding partner of the MRN complex, CtIP, has been implicated in end resection of DNA ends during homology-directed repair (39, 45). The role of MRN in NHEJ has been much less clear. MRX, the yeast orthologue of MRN, functions during NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe (28, 30). In mammalian cells, MRN is not recruited to I-SceI-induced DSBs in G₁, whereas Ku70 is, and MRN does not appear to be required for NHEJ-mediated repair of these DSBs (38, 54). On the other hand, MRN promotes class switch recombination (37) and has been implicated in accurate NHEJ repair during V(D)J recombination (22).The involvement of MRN in ATM signaling and DNA repair pathways has been intriguing from the perspective of telomere biology. While several of the attributes of MRN might be considered a threat to telomere integrity, MRN is known to associate with mammalian telomeres, most likely through an interaction with the TRF2 complex (48, 51, 57). MRN has been implicated in the generation of the telomeric overhang (12), the telomerase pathway (36, 52), the ALT pathway (55), and the protection of telomeres from stochastic deletion events (1). It has also been speculated that MRN may contribute to formation of the t-loop structure (16). t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA (21), are thought to contribute to telomere protection by effectively shielding the chromosome end from DNA damage response factors that interact with DNA ends, including nucleases, and the Ku heterodimer (15).H2AX has been studied extensively in the context of chromosome-internal DSBs. When a DSB is formed, ATM acts near the lesion to phosphorylate a conserved carboxy-terminal serine of H2AX, a histone variant present throughout the genome (7). Phosphorylated H2AX (referred to as γ-H2AX) promotes the spreading of DNA damage factors over several megabases along the damaged chromatin and mediates the amplification of the DNA damage signal (43). The signal amplification is accomplished through a sequence of phospho-specific interactions among γ-H2AX, MDC1, NBS1, RNF8, and RNF168, which results in the additional binding of ATM and additional phosphorylation of H2AX in adjacent chromatin (reviewed in reference 33). The formation of these large domains of altered chromatin, referred to as irradiation-induced foci at DSBs and telomere dysfunction-induced foci (TIFs) at dysfunctional telomeres (44), promotes the binding of several factors implicated in DNA repair, including the BRCA1 A complex and 53BP1 (33).In agreement with a role for H2AX in DNA repair, H2AX-deficient cells exhibit elevated levels of irradiation-induced chromosome abnormalities (5, 9). In addition, H2AX-null B cells are prone to chromosome breaks and translocations in the immunoglobulin locus, indicative of impaired class switch recombination, a process that involves the repair of DSBs through the NHEJ pathway (9, 20). Since H2AX is dispensable for the activation of irradiation-induced checkpoints (8), these data argue that H2AX contributes directly to DNA repair. However, a different set of studies has concluded that H2AX is not required for NHEJ during V(D)J recombination (5, 9) but that it plays a role in homology-directed repair (53). In this study, we have further queried the contribution of H2AX to NHEJ in the context of dysfunctional telomeres.Our aim was to dissect the contribution of MRN and H2AX to DNA damage signaling and NHEJ-mediated repair in response to telomere dysfunction elicited by deletion of TRF2. Importantly, since ATM is the only kinase activated in this setting, deletion of TRF2 can illuminate the specific contribution of these factors in the absence of the confounding effects of ATR signaling (26). This approach revealed a dual role for MRN at telomeres, involving both its function as a sensor in the ATM pathway and its ability to protect telomeres from NHEJ under certain circumstances.     

Rad50 Is Dispensable for the Maintenance and Viability of Postmitotic Tissues

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.The Mre11 complex regulates both DNA damage checkpoint function and repair. Its checkpoint functions appear to be primarily related to its role as a DNA double-strand break (DSB) sensor which binds DNA damage and activates ATM (ataxia-telangiectasia [AT] mutated). The ATM kinase transduces the damage signal via phosphorylating mediators of the damage response (30, 42), which promotes cell cycle arrest, DNA repair, and apoptosis. Mre11 complex functions are compromised in the human chromosome instability syndromes Nijmegen breakage syndrome and AT-like disorder, which are caused by hypomorphic mutations in Nbs1 and Mre11. Cells derived from patients and from mouse models of these diseases exhibit spontaneous DNA damage, ionizing radiation (IR) sensitivity, and checkpoint defects (25, 27, 48, 52, 57).The complex''s primary role in DNA repair is in recombinational DSB repair, and this role likely underlies its essential nature. In Saccharomyces cerevisiae, the complex governs homologous recombination (HR) and nonhomologous end joining (NHEJ) (19), whereas in vertebrate systems it primarily functions in HR (51, 61, 62). In fact, studies of Nbs1-deficient cells suggest that the Mre11 complex may inhibit NHEJ in mammals (62). Data from several species also implicate the Mre11 nuclease in the metabolism of topoisomerase adducts (40, 43, 49). This highly conserved function could also explain why the Mre11 complex is essential.The Mre11 complex''s function at telomeres may also be required for viability. Telomeres protect the ends of linear chromosomes from being recognized as DSBs and thereby activating the DNA damage response (DDR) (9). In S. cerevisiae the Mre11 complex influences telomere length maintenance (5, 28), whereas in mammals the complex interacts with the telomere binding protein Trf2 and localizes to telomeres (63). Loss of Trf2 results in telomere uncapping, causing activation of the DDR, telomere fusions, and senescence (7). Given the association of Mre11 with Trf2, it is conceivable that acute Mre11 complex deficiency in the mouse would phenocopy Trf2 loss and similarly lead to cell death as a result of telomere uncapping.Conclusions regarding the essential nature of HR in general (33, 47, 53) and the Mre11 complex specifically (10, 17, 45, 59, 62) have been derived from the analysis of proliferating cells in vitro or in vivo. The coincidence of DNA replication and the formation of spontaneous DSBs prompted us to test whether the Mre11 complex and, by extension, HR would be essential in quiescent or postmitotic tissues in which the frequency of spontaneous DSBs is significantly reduced. To examine this issue, we generated mice containing a conditional Rad50 allele in which the Rad50 gene could be inactivated in quiescent and postmitotic cells.Our results indicate that Rad50 is not required for homeostasis or viability of quiescent hepatocytes of the adult liver; nor does it appear to be required for maintenance of postmitotic Purkinje cells of the cerebellum. In contrast, Rad50 was required for viability of proliferating tissue culture and bone marrow cells. Rad50-deficient hepatocytes that were induced to divide via hepatectomy were able to achieve limited division and survived despite the presence of DNA damage that persisted long after the bulk of regeneration was complete. Rad50-deficient cells did not exhibit overtly dysfunctional telomeres, suggesting that their loss of viability was not due to acute telomere failure. These data indicate that the Mre11 complex and, by extension, HR may be dispensable in postmitotic cells and are consistent with the interpretation that the replication-associated functions of the Mre11 complex account for its essential nature.     

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.DNA replication must be highly coordinated with chromatin assembly and cell division for correct propagation of genetic information and cell survival. Errors arising during DNA replication are corrected through the functions of numerous pathways including checkpoints and a diversity of DNA repair mechanisms (32, 33, 35). However, in the absence of these critical cellular responses, replication errors can lead to the accumulation of mutations and gross chromosomal rearrangements (GCRs) as well as chromosome loss, a condition generally termed genomic instability (33). Genome instability is a hallmark of many cancers as well as other human diseases (24). There are many mechanisms by which GCRs can arise, and over the last few years numerous genes and pathways have been implicated in playing a role in the suppression of GCRs in Saccharomyces cerevisiae and in some cases in the etiology of cancer (27, 28, 33, 39-47, 51, 53, 56, 58, 60), including S. cerevisiae ASF1, which encodes the main subunit of the replication coupling assembly factor (37, 62).Asf1 is involved in the deposition of histones H3 and H4 onto newly synthesized DNA during DNA replication and repair (62), and correspondingly, asf1 mutants are sensitive to chronic treatment with DNA-damaging agents (2, 30, 62). However, asf1 mutants do not appear to be repair defective and can recover from acute treatment with at least some DNA-damaging agents (2, 8, 30, 31, 54), properties similar to those described for rad9 mutants (68). In the absence of Asf1, both the DNA damage and replication checkpoints become activated during normal cell growth, and in the absence of checkpoint execution, there is a further increase in checkpoint activation in asf1 mutants (30, 46, 54). It has been suggested that asf1 mutants are defective for checkpoint shutoff and that this might account for the increased steady-state levels of checkpoint activation seen in asf1 mutants (8); however, another study has shown that asf1 mutants are not defective for checkpoint shutoff and that in fact Asf1 and the chromatin assembly factor I (CAF-I) complex act redundantly or cooperate in checkpoint shutoff (31). Furthermore, Asf1 might be involved in proper activation of the Rad53 checkpoint protein, as Asf1 physically interacts with Rad53 and this interaction is abrogated in response to exogenous DNA damage (15, 26); however, the physiological relevance of this interaction is unclear. Asf1 is also required for K56 acetylation of histone H3 by Rtt109, and both rtt109 mutants and histone H3 variants that cannot be acetylated (38) share many of the properties of asf1 mutants, suggesting that at least some of the requirement for Asf1 in response to DNA damage is mediated through Rtt109 (11, 14, 22, 61). Subsequent studies of checkpoint activation in asf1 mutants have led to the hypothesis that replication coupling assembly factor defects result in destabilization of replication forks which are then recognized by the replication checkpoint and stabilized, suggesting that the destabilized replication forks account for both the increased GCRs and increased checkpoint activation seen in asf1 mutants (30). This hypothesis is supported by other recent studies implicating Asf1 in the processing of stalled replication forks (16, 57). This role appears to be independent of CAF-I, which can cooperate with Asf1 in chromatin assembly (63). Asf1 has also been shown to function in disassembly of chromatin, suggesting other possibilities for the mechanism of action of Asf1 at the replication fork (1, 2, 34). Thus, while Asf1 is thought to be involved in progression of the replication fork, both the mechanism of action and the factors that cooperate with Asf1 in this process remain obscure.Stalled replication forks, particularly those that stall at sites of DNA damage, can be processed by homologous recombination (HR) (6) or by a mechanism known as postreplication repair (PRR) (reviewed in reference 67). There are two PRR pathways, an error-prone pathway involving translesion synthesis (TLS) by lower-fidelity polymerases and an error-free pathway thought to involve template switching (TS) (67). In S. cerevisiae, the PRR pathways are under the control of the RAD6 epistasis group (64). The error-prone pathway depends on monoubiquitination of proliferating cell nuclear antigen (PCNA) on K164 by Rad6 (an E2 ubiquitin-conjugating enzyme) by Rad18 (E3 ubiquitin ligase) (23). This results in replacement of the replicative DNA polymerase with nonessential TLS DNA polymerases, such as REV3/REV7-encoded DNA polymerase ζ (polζ) and RAD30-encoded DNA polη, which can bypass different types of replication-blocking damage (67). The error-free pathway is controlled by Rad5 (E3) and a complex consisting of Ubc13 and Mms2 (E2 and E2 variant, respectively), which add a K63-linked polyubiquitin chain to monoubiquitinated PCNA, leading to TS to the undamaged nascent sister chromatid (4, 25, 65). Furthermore, in addition to modification with ubiquitin, K164 of PCNA can also be sumoylated by Siz1, resulting in subsequent recruitment of the Srs2 helicase and inhibition of deleterious Rad51-dependent recombination events (50, 52, 55), although it is currently unclear if these are competing PCNA modifications or if both can exist on different subunits in the same PCNA trimer. A separate branch of the Rad6 pathway involving the E3 ligase Bre1 monoubiquitinates the histone H2B (29, 69) as well as Swd2 (66), which stimulates Set1-dependent methylation of K4 and Dot1-dependent methylation of K79 of histone H3 (48, 49, 66). Subsequently, K79-methylated H3 recruits Rad9 and activates the Rad53 checkpoint (19, 70). Activation of Rad53 is also bolstered by Rad6-Rad18-dependent ubiquitination of Rad17, which is part of the 9-1-1 complex that functions upstream in the checkpoint pathway (17). Finally, Rad6 complexes with the E3 Ubr1, which mediates protein degradation by the N-end rule pathway (13).Due to the role of the PRR pathways at stalled replication forks and a recent study implicating the Rad6 pathway in the suppression of GCRs (39), we examined the relationship between these ubiquitination and sumoylation pathways and the Asf1 pathway in order to gain additional insights into the function of Asf1 during DNA replication and repair. Our findings suggest that Asf1 has multiple functions that prevent replication damage or act in the cellular responses to replication damage and that these functions are modified by and interact with the PRR pathways. The TLS PRR pathway does not appear to be involved, and both a Dun1-dependent replication checkpoint and HR are important for preventing the deleterious effects of PRR and Asf1 pathway defects. We hypothesize that this newly observed cooperation between Asf1 and the PRR pathways may be required for resolving stalled replication forks, leading to suppression of GCRs and successful DNA replication.     

Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.Homologous recombination (HR) is often described as a double-edged sword: it can maintain genome stability by promoting DNA repair, while its injudicious action can disturb genome stability by causing gross chromosome rearrangement (GCR) or loss of heterozygosity (LOH). Both GCR and LOH are potential precursors of diseases such as cancer, and consequently there is need to control when and how HR is used.A key step in most HR is the loading of the Rad51 recombinase onto single-stranded DNA (ssDNA), which forms a nucleoprotein filament (nucleofilament) that catalyzes the pairing of homologous DNAs and subsequent strand invasion (32). This is a critical point at which recombination can be regulated through the removal of the Rad51 filament (60). Early removal can prevent strand invasion altogether, freeing the DNA for alternative processing. Later removal may limit unnecessary filament growth, free the 3′-OH of the invading strand to prime DNA synthesis, and ultimately enable ejection of the invading strand, which is important for the repair of double-strand breaks (DSBs) by synthesis-dependent strand annealing (SDSA). SDSA avoids the formation of Holliday junctions that can be resolved into reciprocal exchange products (crossovers), which may result in GCR or LOH if the recombination is ectopic or allelic, respectively.One enzyme that appears to be able to control Rad51 in the aforementioned manner is the yeast superfamily 1 DNA helicase Srs2 (42). In Saccharomyces cerevisiae, Srs2 is recruited to stalled replication forks by the SUMOylation of PCNA, and there it appears to block Rad51-dependent HR in favor of Rad6- and Rad18-dependent postreplication repair (1, 2, 35, 50, 53, 58). In vitro Srs2 can strip Rad51 from ssDNA via its DNA translocase activity (31, 62) and therefore probably controls HR at stalled replication forks by acting as a Rad51 disruptase. In accord with this, chromatin immunoprecipitation analysis has shown that Rad51 is enriched at or near replication forks in an srs2 mutant (50). Srs2 also plays an important role in crossover avoidance during DSB repair, where it is thought to promote SDSA by both disrupting Rad51 nucleofilaments and dissociating displacement (D) loops (20, 27).Srs2 is conserved in the fission yeast Schizosaccharomyces pombe (19, 43, 63) and has a close relative in bacteria called UvrD, which can similarly control HR by disrupting RecA nucleofilaments (61). However, an obvious homologue in mammals has not been detected. Recently, two mammalian members of the RecQ DNA helicase family, BLM and RECQL5, were shown to disrupt Rad51 nucleofilaments in vitro (11, 25), although in the case of BLM, this activity appears to be relatively weak (5, 55). Nevertheless these data have led to speculation that both BLM and RECQL5 might perform a function similar to that of Srs2 in vivo (6). Certainly mutational inactivation of either helicase results in elevated levels of HR and genome instability, with an associated increased rate of cancer (23, 25). However, BLM and RECQL5 are not the only potential Rad51 disruptases in mammals; a relative of Srs2 and UvrD called FBH1 was recently implicated in this role by genetic studies of its orthologue in S. pombe and by its ability to partially compensate for the loss of Srs2 in S. cerevisiae, which, unlike S. pombe, lacks an FBH1 orthologue (15). FBH1 is so named because of an F box near its N terminus—a feature that makes it unique among DNA helicases (28). The F box is important for its interaction with SKP1 and therefore the formation of an E3 ubiquitin ligase SCF (SKP1-Cul1-F-box protein) complex (29). The targets of this complex are currently unknown. In S. pombe, mutations within Fbh1''s F-box block interaction with Skp1 and prevent Fbh1 from localizing to the nucleus and forming damage-induced foci therein (57). Fbh1''s role in constraining Rad51 activity in S. pombe is evidenced by the increase in spontaneous Rad51 foci and accumulation of UV irradiation-induced Rad51-dependent recombination intermediates in an fbh1Δ mutant (47). Moreover, loss of both Fbh1 and Srs2 in S. pombe results in a synergistic reduction in cell viability, and like Srs2, Fbh1 is essential for viability in the absence of the S. pombe RecQ family DNA helicase Rqh1, which processes recombination intermediates (47, 48). In both cases the synthetic interaction is suppressed by deleting rad51, suggesting that Fbh1 works in parallel with Srs2 and Rqh1 to prevent the formation of toxic recombination intermediates. In yeast, Rad51-mediated recombination is dependent on Rad52 (Rad22 in S. pombe), which is believed to promote the nucleation of Rad51 onto DNA that is coated with the ssDNA binding protein replication protein A (RPA) (18, 32). Intriguingly, the genotoxin sensitivity and recombination deficiency of a rad22 mutant are suppressed in a Rad51-dependent manner by deleting fbh1 (48). This suggests that Fbh1 and Rad22 act in opposing ways to modulate the assembly of the Rad51 nucleofilament. Although current data indicate a role for Fbh1 in controlling HR, the only evidence so far that Fbh1 limits recombinant formation is in chicken DT40 cells, for which a modest increase in sister chromatid exchange has been noted when FBH1 is deleted (30).Here we present in vivo evidence suggesting that Fbh1 does indeed act as a Rad51 disruptase, which is dependent on its DNA helicase/translocase activity. We confirm predictions that this activity works in opposition to Rad22 for the loading of Rad51 onto DNA and show that Fbh1''s modulation of Rad51 activity, while not essential for limiting spontaneous direct-repeat recombination, is critical for preventing recombination at blocked replication forks. Finally, we highlight similarities and differences between Fbh1 and Srs2, based on their mutant phenotypes and relative abilities to suppress recombination when overexpressed. Overall our data affirm that Fbh1 is one of the principal modulators of Rad51 activity in fission yeast and therefore may play a similar role in vertebrates.     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6, 14, 21, 32, 38, 41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G₁/S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13, 15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNA-damaging agents, have been suggested (33, 54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5′ ends of the DSB to generate 3′ ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4). RPA and members of the RAD52 epistasis group of gene products, such as Rad51, Rad52, and Rad54, bind to the resulting 3′ ssDNA strands and form a helical, nucleoprotein filament that facilitates the invasion of a damaged DNA strand into the homologous double-stranded DNA partner. The human Rad51 protein is a structural and functional homolog of the Escherichia coli RecA protein, which promotes homologous pairing and strand transfer reactions in vitro. Both Rad51 and Rad52 bind specifically to the terminal regions of tailed duplex DNA, the substrate thought to initiate recombination in vivo. Furthermore, nucleoprotein filaments of Rad51, formed on tailed DNA, catalyze strand invasion of homologous duplex DNA in a reaction that is stimulated by Rad52 and RPA (3).Epstein-Barr virus (EBV) is a human herpesvirus that infects B lymphocytes, inducing their continuous proliferation. In B-lymphoblastoid cell lines, there is no production of virus particles, which is termed latent infection (52). Reactivation from latency is characterized by the expression of lytic genes, and one of the first detectable changes is the expression of the BZLF1 immediate-early gene product, which trans-activates viral promoters (16), leading to an ordered cascade of viral early and late gene expression. This lytic EBV DNA replication occurs in discrete sites in nuclei, called replication compartments, in which seven viral replication proteins are assembled (44). The viral genome is amplified several hundredfold by the viral replication machinery and is thought to generate highly branched replication intermediates through HR coupled with viral DNA replication (48). With the progression of lytic replication, the replication compartments become larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages of infection (8, 45).We previously isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter, leading to a highly efficient induction of lytic replication (28). Using this system, we have demonstrated that the induction of the EBV lytic program results in the inhibition of replication of cellular DNA in spite of the replication of viral DNA (28) and elicits a cellular DNA damage response, with the activation of the ATM-Chk2-p53 DNA damage transduction pathway (29). The DNA damage sensor MRN complex and phosphorylated ATM are recruited and retained in viral replication compartments (29).Here we report that RPA32 is extensively phosphorylated after EBV lytic replication is induced, with the phosphorylation sites in accordance with those for ATM. Phosphorylated RPA, Rad51, and Rad52, which are involved in HR repair (HRR), are recruited and retained in viral replication compartments as well as the MRN complex. Furthermore, DSBs could be demonstrated to occur during viral genome synthesis in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference (RNAi) knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that HR and/or repair of viral DNA genome might occur, coupled with DNA replication, to facilitate viral genome synthesis.     

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.     

Widely Conserved Recombination Patterns among Single-Stranded DNA Viruses

The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.Genetic recombination is a ubiquitous biological process that is both central to DNA repair pathways (10, 57) and an important evolutionary mechanism. By generating novel combinations of preexisting nucleotide polymorphisms, recombination can potentially accelerate evolution by increasing the population-wide genetic diversity upon which adaptive selection relies. Recombination can paradoxically also prevent the progressive accumulation of harmful mutations within individual genomes (18, 35, 53). Whereas its ability to defend high-fitness genomes from mutational decay possibly underlies the evolutionary value of sexuality in higher organisms, in many microbial species where pseudosexual genetic exchange is permissible among even highly divergent genomes, recombination can enable access to evolutionary innovations that would otherwise be inaccessible by mutation alone.Such interspecies recombination is fairly common in many virus families (8, 17, 27, 44, 82). It is becoming clear, however, that as with mutation events, most recombination events between distantly related genomes are maladaptive (5, 13, 38, 50, 63, 80). As genetic distances between parental genomes increase, so too does the probability of fitness defects in their recombinant offspring (16, 51). The viability of recombinants is apparently largely dependent on how severely recombination disrupts coevolved intragenome interaction networks (16, 32, 51). These networks include interacting nucleotide sequences that form secondary structures, sequence-specific protein-DNA interactions, interprotein interactions, and amino acid-amino acid interactions within protein three-dimensional folds.One virus family where such interaction networks appear to have a large impact on patterns of natural interspecies recombination are the single-stranded DNA (ssDNA) geminiviruses. As with other ssDNA viruses, recombination is very common among the species of this family (62, 84). Partially conserved recombination hot and cold spots have been detected in different genera (39, 81) and are apparently caused by both differential mechanistic predispositions of genome regions to recombination and natural selection disfavoring the survival of recombinants with disrupted intragenome interaction networks (38, 51).Genome organization and rolling circle replication (RCR)—the mechanism by which geminiviruses and many other ssDNA viruses replicate (9, 67, 79; see reference 24 for a review)—seem to have a large influence on basal recombination rates in different parts of geminivirus genomes (20, 33, 39, 61, 81). To initiate RCR, virion-strand ssDNA molecules are converted by host-mediated pathways into double-stranded “replicative-form” (RF) DNAs (34, 67). Initiated by a virus-encoded replication-associated protein (Rep) at a well-defined virion-strand replication origin (v-ori), new virion strands are synthesized on the complementary strand of RF DNAs (28, 73, 74) by host DNA polymerases. Virion-strand replication is concomitant with the displacement of old virion strands, which, once complete, yields covalently closed ssDNA molecules which are either encapsidated or converted into additional RF DNAs. Genome-wide basal recombination rates in ssDNA viruses are probably strongly influenced by the specific characteristics of host DNA polymerases that enable RCR. Interruption of RCR has been implicated directly in geminivirus recombination (40) and is most likely responsible for increased basal recombination rates both within genes transcribed in the opposite direction from that of virion-strand replication (40, 71) and at the v-ori (1, 9, 20, 69, 74).Whereas most ssDNA virus families replicate via either a rolling circle mechanism (the Nanoviridae, Microviridae, and Geminiviridae) (3, 23, 24, 31, 59, 67, 74) or a related rolling hairpin mechanism (the Parvoviridae) (25, 76), among the Circoviridae only the Circovirus genus is known to use RCR (45). Although the Gyrovirus genus (the other member of the Circoviridae) and the anelloviruses (a currently unclassified ssDNA virus group) might also use RCR, it is currently unknown whether they do or not (78). Additionally, some members of the Begomovirus genus of the Geminiviridae either have a second genome component, called DNA-B, or are associated with satellite ssDNA molecules called DNA-1 and DNA-Beta, all of which also replicate by RCR (1, 47, 68).Recombination is known to occur in the parvoviruses (19, 43, 70), microviruses (66), anelloviruses (40, 46), circoviruses (11, 26, 60), nanoviruses (30), geminivirus DNA-B components, and geminivirus satellite molecules (2, 62). Given that most, if not all, of these ssDNA replicons are evolutionarily related to and share many biological features with the geminiviruses (22, 31, 36), it is of interest to determine whether conserved recombination patterns observed in the geminiviruses (61, 81) are evident in these other groups. To date, no comparative analyses have ever been performed with different ssDNA virus families to identify, for example, possible influences of genome organization on recombination breakpoint distributions found in these viruses.Here we compare recombination frequencies and recombination breakpoint distributions in most currently described ssDNA viruses and satellite molecules and identify a number of sequence exchange patterns that are broadly conserved across this entire group.     

Effect of Telomere Proximity on Telomere Position Effect,Chromosome Healing,and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.Telomeres are composed of a six-base pair repeat sequence and associated proteins that together form a cap to protect the ends of chromosomes and prevent chromosome fusion (6). Telomeres are actively maintained by the enzyme telomerase in human germ line cells but shorten with age in most somatic cells due to the low level of expression of telomerase (12). When a telomere shortens to the point that it is recognized as a double-strand break (DSB), it serves as a signal for replicative cell senescence (13). Human cells that lose the ability to senesce continue to show telomere shortening and eventually enter crisis, which involves increased chromosome fusion, aneuploidy, and cell death (11, 15). An important step that is required for continued division of cancer cells is therefore that they possess the ability to maintain telomeres, not only to avoid senescence but also to avoid chromosome fusion brought on by crisis (11, 25).In addition to their role in protecting the ends of chromosomes, telomeres can also inhibit the expression of nearby genes, called telomere position effect (TPE). TPE has been proposed to have a role in the cellular response to changes in telomere length (26); however, the function of TPE remains unknown. TPE has been extensively studied in Saccharomyces cerevisiae using transgenes integrated near telomeres on truncated chromosomes (1, 2, 22, 47). These studies demonstrated that TPE involves changes in chromatin conformation and is dependent upon both the distance from the telomere and telomere length (55). Subsequent studies of endogenous yeast genes, however, revealed that the influence of TPE on gene expression varies depending on the presence of insulator sequences (18, 45). TPE also occurs in mammalian cells and has been implicated in the loss of expression of genes relocated near telomeres in a variety of human syndromes (9, 16, 28, 58, 59). As in yeast, transgenes located near telomeres have been used to study TPE in the C33-A (32) and HeLa (4) human cervical carcinoma cell lines. We have also studied TPE using transgenes located adjacent to telomeres in mouse embryonic stem (ES) cells, mouse embryo fibroblasts, and transgenic mice (43). However, none of the studies of TPE in mammalian cells has addressed the distance over which TPE extends from the telomere, and so the number of genes whose expression is likely to be affected is not known.The presence of a telomere can also influence the sensitivity of subtelomeric regions to DSBs. We previously demonstrated the sensitivity of subtelomeric regions to DSBs using selectable transgenes and a recognition site for the I-SceI endonuclease that are integrated immediately adjacent to a telomere. Unlike I-SceI-induced DSBs at most locations, which primarily result in small deletions (27, 34, 46, 50), I-SceI-induced DSBs near telomeres commonly result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability in both mouse ES cells (37) and human tumor cells (65). Therefore, depending on the size of the sensitive region, the combined targets of the subtelomeric regions on all telomeres could contribute significantly to the genomic instability caused by ionizing radiation or other agents that produce DSBs (35). This sensitivity to DSBs may result from a deficiency in DSB repair since regions near telomeres in yeast are deficient in nonhomologous end joining, resulting in an increase in GCRs (48). One possible reason for a deficiency in DSB repair near telomeres is the role of the telomere in preventing chromosome fusion. Telomeric repeat sequences in yeast have been shown to suppress the activation of cell cycle checkpoints in response to DSBs (39). Similarly, the human TRF2 protein, which is required to prevent chromosome fusion, has been demonstrated to inhibit ATM (31), whose activation is instrumental in the repair of DSBs in heterochromatin (20).One mechanism for avoiding the consequences of DSBs near telomeres is through the addition of a new telomere to the site of a DSB, termed chromosome healing (44). Studies in yeast have shown that chromosome healing occurs through the de novo addition of telomeric repeat sequences by telomerase (14, 33, 38). Chromosome healing in S. cerevisiae is inhibited by the 5′-3′ helicase, Pif1 (52), with Pif1-deficient cells showing up to a 1,000-fold increase in chromosome healing (33, 38). The ability of Pif1 to inhibit chromosome healing has been proposed to serve as a mechanism to prevent chromosome healing from interfering with DSB repair (63). Mammalian cells that express telomerase are also capable of performing chromosome healing. We have shown that chromosome healing can also occur following spontaneous telomere loss (17, 49) or DSBs near telomeres in a human cancer cell line (65) or mouse ES cells (19, 54). We have also shown that chromosome healing can prevent the chromosome instability resulting from DSBs near telomeres (19). Because the de novo addition of telomeric repeat sequences has not been observed in mammalian cells at I-SceI-induced DSBs at interstitial sites (27, 34, 46, 50), we have proposed that chromosome healing is inhibited at most locations but serves as an important mechanism for dealing with DSBs near telomeres that would otherwise result in chromosome instability. However, an alternative possibility that has not been ruled out is that chromosome healing also occurs at interstitial sites but that the large terminal deletions that it causes at these sites results in cell death.In the present study, we address several key questions regarding the importance of telomere proximity on TPE, chromosome healing, and sensitivity to DSBs by investigating how telomere proximity affects these processes. The first of these questions involves establishing the distance over which TPE extends from the telomere to gain insights into the numbers of genes that would be affected by changes in TPE. Second, we will investigate whether chromosome healing can occur at a site that is distant from a telomere but in which terminal deletions are known not to be lethal. This will determine for the first time whether chromosome healing is limited to regions near telomeres. Finally, we will investigate the size of the region near a telomere that is sensitive to DSBs, which will address the potential importance of the subtelomeric region as a target for ionizing radiation-induced genomic instability (35). The distance over which a telomere can exert its effects was investigated by comparing TPE, chromosome healing, and the sensitivity to DSBs at a site 100 kb from a telomere with a site immediately adjacent to the same telomere. As a control for the efficiency of generating DSBs at these sites, we have also analyzed the frequency of small deletions, the most common type of I-SceI-induced DNA rearrangement at interstitial sites in mammalian cells (27, 60). Small deletions serve as an excellent internal control for comparing the frequency of other types of rearrangements since we have previously observed a similar frequency of small deletions at telomeric and interstitial sites (65). The results provide important information on the distance over which a telomere can influence TPE, chromosome healing, and the sensitivity to DSBs.     

Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup

Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault''s rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault''s rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault''s rousette.Coronaviruses infect a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterization, coronaviruses were traditionally classified into three distinct groups, groups 1, 2, and 3 (3, 27, 59). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has renamed the traditional group 1, 2, and 3 coronaviruses as Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx). Coronaviruses are known to have a high frequency of recombination as a result of their unique mechanism of viral replication (27). Such tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (24, 47, 52).The severe acute respiratory syndrome (SARS) epidemic has boosted interest in the study of coronaviruses in humans and animals (21, 34, 38, 41, 54). In the past few years, there has been a dramatic increase in the number of newly described human and animal coronaviruses (2, 4, 5, 8-10, 15-20, 23, 25, 28, 30, 32, 35, 36, 39, 43, 45, 50, 51, 53, 56, 58). Two novel human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1), belonging to Alphacoronavirus and Betacoronavirus, respectively, have been discovered, in addition to the human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), and SARS coronavirus (SARS-CoV) (17, 29, 45, 53, 55). We have also previously described the discovery of a diversity of novel coronaviruses in wild bats and birds in China, including SARSr-Rh-BatCoV, belonging to Betacoronavirus subgroup B, from Chinese horseshoe bats (30, 48, 56). Among these novel coronaviruses, three avian coronaviruses were found to belong to a novel subgroup of Gammacoronavirus (Gammacoronavirus subgroup C), while three bat coronaviruses were found to belong to two novel subgroups of Betacoronavirus (Betacoronavirus subgroups C and D) (48, 50). Based on the presence of the huge diversity of coronaviruses in Alphacoronavirus and Betacoronavirus among various bat species, bats are likely the reservoir for the ancestor of these two coronavirus genera (47).During our genome analysis of these novel coronaviruses, one of them, Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), belonging to Betacoronavirus subgroup D, which was identified in Leschenault''s rousette bats, was found to display marked nucleotide and amino acid sequence polymorphism among the four strains with complete genome sequences (50). In our study on HCoV-HKU1, it has been shown that such sequence polymorphisms may indicate the presence of different genotypes (52). By complete genome sequence analysis of the potentially different genotypes of HCoV-HKU1, we have demonstrated for the first time natural recombination in a human coronavirus, resulting in the generation of at least three genotypes (52). We have also recently shown that recombination between different strains of SARSr-Rh-BatCoV from different regions of China may have given rise to the emergence of civet SARSr-CoV (31). To investigate the presence of different genotypes of Ro-BatCoV HKU9, the complete RNA-dependent RNA polymerase (RdRp) (corresponding to nsp12), spike (S), and nucleocapsid (N) gene sequences of Ro-BatCoV HKU9 from 10 additional bats were determined. Since sequence analysis showed the possible coexistence of different genotypes in seven bat individuals, complete genome sequencing of these distinct genotypes from two bats was carried out to investigate for possible recombination events among the different genotypes. In addition, serological characterization of Ro-BatCoV HKU9 was also performed by Western blot and enzyme immunoassays using recombinant Ro-BatCoV HKU9 nucleocapsid proteins and recombinant nucleocapsid proteins of Betacoronavirus subgroup A, B, and C coronaviruses to determine possible cross-reactivity among the different Betacoronavirus subgroups and the seroepidemiology of Ro-BatCoV HKU9 in Leschenault''s rousette bats.