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.     

The Direct Binding of Mrc1, a Checkpoint Mediator,to Mcm6, a Replication Helicase,Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.Progression of the DNA replication machinery along chromosomes is a complex process. Replication forks pause occasionally when they encounter genomic regions that are difficult to replicate, such as highly transcribed regions, tRNA genes, and regions with specialized chromatin structure, like centromeric and heterochromatic regions (17). Replication forks also stall when treated with chemicals like methyl methanesulfonate (MMS), which causes DNA damage, or hydroxyurea (HU), which limits the cellular concentration of the deoxynucleoside triphosphate pool (17). Because de novo assembly and programming of the replisome do not occur after the onset of S phase (18), DNA replication forks must be protected from replicative stresses. The DNA replication checkpoint constitutes a surveillance mechanism for S-phase progression that safeguards replication forks from various replicative stresses (22, 38, 40), and malfunction of this checkpoint leads to chromosome instability and cancer development in higher organisms (4, 9).The Saccharomyces cerevisiae DNA replication checkpoint mediator Mrc1 is functionally conserved and is involved directly in DNA replication as a component of the replisome (1, 8, 16, 19, 29, 30). Mrc1, together with Tof1 and Csm3, is required for forming a replication pausing complex when the fork is exposed to replicative stress by HU (16). The pausing complex subsequently triggers events leading to DNA replication checkpoint activation and hence stable replicative arrest. A sensor kinase complex, Mec1-Ddc2 (ATR-ATRIP homolog of higher eukaryotes), is then recruited to the complex (14, 16). Mec1-Ddc2-mediated phosphorylation of Mrc1 activates the pausing complex, and phosphorylated Mrc1 likely recruits Rad53 (a putative homolog of CHK2 of higher eukaryotes), which is then activated via phosphorylation by Mec1-Ddc2 (1, 16, 20, 30). Activated Rad53 subsequently elicits a stress responses, i.e., stabilization of replication forks, induction of repair genes, and suppression of late-firing origins (24). It remains unclear, however, whether DNA replication checkpoint activation is induced in response to DNA damage by MMS, a reagent commonly used to study the DNA replication stress response. Several lines of evidence have suggested that MMS-induced damage is also sensed directly by the replication machinery (38, 40).Although biochemical and genetic interaction data have placed Mrc1 at the center of the replication checkpoint signal transduction cascade, its molecular function remains largely unknown. The proteins Mrc1, Tof1, and Csm3 associate with the Mcm complex (8, 27), a heterohexameric DNA helicase consisting of Mcm2 to Mcm7 proteins which unwinds the parental DNA duplex to allow replisome progression (3, 12, 18, 31, 32, 35). The Mcm complex associates with a specific set of regulatory proteins at forks to form replisome progression complexes (8). In addition to Mcm, Tof1, Csm3, and Mrc1, replisome progression complexes include factors such as Cdc45 and the GINS complex that are also required for fork progression (13, 26, 31, 32, 39). Claspin, a putative Xenopus laevis homolog of Mrc1, is also reported to associate with Cdc45, DNA polymerase ɛ (Polɛ), replication protein A, and two of the replication factor C complexes in aphidicolin-treated Xenopus egg extracts (19). Recently, Mrc1 was reported to interact directly with Polɛ (23).The aim of this study was to provide mechanistic insight into Mrc1 function in the DNA replication checkpoint. For this purpose, it was essential to identify, among all the essential proteins in the replication machinery, a specific protein that interacts with Mrc1 and to examine the role of this interaction in the DNA replication checkpoint. We found that Mrc1 interacts with Mcm6 directly and specifically. When the interaction between Mrc1 and Mcm6 was impaired, cells no longer activated the DNA replication checkpoint in response to MMS-induced replicative stress. Interestingly and unexpectedly, this interaction was not required for DNA replication checkpoint activation in response to HU-induced replicative stress. Our results provide the first mechanistic evidence that cells use separate mechanisms to transmit replicative stresses caused by MMS and HU for DNA replication checkpoint activation.     

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.     

BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3^ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT₂) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1^ATM and Rad3^ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT₂ domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT₂ γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.Double-strand breaks (DSBs) are among the most dangerous forms of DNA damage (26, 30). Human cells experience DSBs several times a day, either during normal metabolism or as a consequence of exposure to DNA-damaging agents, such as ionizing radiation (IR) (18). Importantly, the unfaithful repair of such breaks can result in genome instability and cancer. The response to DSBs is coordinated by a conserved signal transduction cascade, which leads to cell cycle arrest and activation of DNA repair and constitutes the checkpoint response (9, 14, 20). The essential players in this process fall into four groups: sensors, mediators, transducers, and effectors (20). Sensors are the first to recognize and bind to DNA breaks and include the Mre11-Rad50-Nbs1 complex in humans and Schizosaccharomyces pombe (Mre11-Rad50-Xrs2 in Saccharomyces cerevisiae). The PIKKs (phosphoinositide 3-kinase-like kinases) ATR-ATRIP (ScMec1-ScDdc2/SpRad3-SpRad26) and ATM (ScTel1/SpTel1) act as transducers that transmit the signal to the effector kinases Chk1 (ScChk1/SpChk1) and Chk2 (ScRad53/SpCds1), whose role is to target downstream targets, such as p53 in mammals, and to amplify the signal (9, 14, 20).Signaling between transducers and effectors is facilitated and enhanced by mediator proteins (19, 20). In the fission yeast Schizosaccharomyces pombe, Crb2/Rhp9 is a critical mediator of the DNA damage checkpoint (31, 42) and is related to Saccharomyces cerevisiae Rad9 and mammalian 53BP1 (p53 binding protein 1). Rad3^ATR-Rad26^ATRIP phosphorylates Crb2 in response to damage, and Crb2 is required for phosphorylation of Chk1 by Rad3^ATR-Rad26^ATRIP (31). Chk1, in turn, restrains entry into mitosis by phosphorylating and thus inactivating the phosphatase Cdc25 that is a mitotic inducer (10, 11, 28). Crb2-null cells are sensitive to a range of genotoxins and are unable to delay division in response to DNA damage (31, 42).Crb2 is a nuclear protein that rapidly relocalizes to DSBs. This occurs on such a large scale that IR-induced nuclear foci (IRIF) of yellow fluorescent protein (YFP)-tagged Crb2 expressed from the endogenous promoter are readily detected by live cell microscopy (5). These foci colocalize with homologous recombination (HR) repair factors such as Rad22^Rad52. Two types of histone modifications regulate Crb2 localization at DSBs: C-terminal phosphorylation of histone H2A, denoted as γ-H2A (23), and lysine-20 dimethylation of histone H4, denoted as H4-K20me2 (32). Phosphorylation of an SQ motif within the C-terminal tail of histone H2A of budding yeast or fission yeast, or the H2AX variant in mammals, is one of the earliest cellular responses triggered by DNA damage (3, 23, 29). The γ-H2A/X modification, which is catalyzed by the checkpoint kinases ATR^Rad3 and ATM^Tel1, spans large distances on both sides of a DSB, and it plays a critical role in recruiting DNA damage response proteins, chromatin remodeling complexes, and cohesin (2, 21, 23, 34, 35, 37, 38, 40). Protein crystallography and biochemical studies established that mammalian Mdc1, S. pombe Crb2, and Brc1 DNA damage response proteins directly bind the phosphorylated tail of histone H2A/X through tandem C-terminal BRCT domains (16, 35, 40). In contrast to γ-H2A, H4-K20 methylation catalyzed by Set9/Kmt5 histone methyltransferase appears to be constitutive and not regulated by DNA damage (32). H4-K20me2 directly binds tandem tudor domains (Tudor₂) located to the N-terminal side of the BRCT domains in Crb2 (1).YFP-Crb2 does not form IRIF in hta1-S129A hta2-S128A (htaAQ) or rad3Δ tel1Δ cells, in which γ-H2A phosphorylation is abolished (23), or in set9Δ cells or tudor domain mutants of Crb2 that ablate binding to H4-K20me2 (6, 32). However, Crb2 checkpoint functions are only partially impaired in an htaAQ set9Δ strain, implying that physiologically significant recruitment of Crb2 to DSBs also occurs by a histone modification-independent pathway. Indeed, we found that YFP-Crb2 forms microscopically visible foci in htaAQ set9Δ cells when DSBs are created by HO endonuclease or by treating cells in G₁ phase with IR (6). Unlike IR-induced DSBs formed during G₂ phase, these types of DSBs lack an intact sister chromatid that can be used for HR repair and therefore they are highly persistent. Further analysis revealed that the histone modification-independent pathway of recruiting Crb2 to DSBs requires threonine-215 (Thr215) phosphorylation catalyzed by the cyclin-dependent kinase (CDK) Cdc2, which facilitates an interaction with Cut5 (ScDpb11; mammalian TopBP1) (6, 8, 31). The crb2-T215A mutation does not ablate YFP-Crb2 IRIF formation; however, Crb2 Thr215 phosphorylation is required for formation of YFP-Crb2 foci at persistent DSBs in htaAQ or set9Δ cells, and combining crb2-T215A with htaAQ or set9Δ abolishes Crb2 function (6).The tandem C-terminal BRCT domains (BRCT₂) of Crb2 not only mediate interactions with γ-H2A but also coordinate Crb2 homodimerization (4). In fact, replacing BRCT₂ with a leucine zipper (LZ) dimerization motif restores substantial function to Crb2 without restoring its ability to form IRIF. Thus, the most crucial task of the Crb2 BRCT domains is to provide a homodimerization platform, while binding to γ-H2A provides an additional function that is necessary for full resistance to DNA damage (4).In a recent study, Kilkenny et al. (16) solved the crystal structures of Crb2-BRCT₂ alone and in complex with a γ-H2A-derived phosphopeptide containing the common C-terminal residues of H2A.1 and H2A.2 (the two H2A paralogues in S. pombe). These analyses revealed the structural determinants of BRCT₂ binding to γ-H2A and BRCT₂-mediated homodimerization of Crb2. Ser666 was found to be critical for homodimerization in vitro, and mutation of this residue severely impaired Crb2 function in vivo. Residues Ser548 and Lys619 were identified as important for the interaction with the phosphate group on γ-H2A.1 pSer129. However, a charge reversal mutation of Lys619 did not abrogate Crb2 IRIF formation measured using methanol-fixed cells, although it did disrupt binding to a γ-H2A peptide in vitro (16). These unexpected findings indicated that γ-H2A likely has an indirect role in regulating Crb2 localization at DSBs. Here, we investigate Crb2 localization in live cells and find that while mutations of Ser548 or Lys619 partially impair Crb2 IRIF, the corresponding double mutant is severely deficient in Crb2 IRIF formation. Our findings and an independent study by Sanders et al. (33) show that γ-H2A binding to BRCT₂ is critical for Crb2 focus formation at IR-induced DSBs and for maintaining a DNA damage checkpoint response.     

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G₁) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.In response to low levels of the DNA-alkylating agent methyl methanesulfonate (MMS), wild-type yeast cells slow their progression through S phase, while cells lacking the appropriate upstream checkpoint kinase (Mec1 in the budding yeast Saccharomyces cerevisiae; Rad3 in the fission yeast Schizosaccharomyces pombe) or the appropriate downstream checkpoint kinase (Rad53 in budding yeast, Cds1 in fission yeast) fail to do so. Other DNA-damaging agents also cause a checkpoint-dependent slowing of S phase, in vertebrates as well as in yeasts. This slowing of S phase in response to DNA damage is sometimes called the “intra-S-phase” checkpoint (3, 6, 22, 23, 26, 28, 36, 37, 45, 53). Here we shall refer to it as the “S-phase damage” checkpoint.Prior to this report, the downstream portions of the checkpoint pathway(s) that slow S phase in response to DNA damage in fission yeast were unclear. However, the upstream portions of these pathways in fission yeast and other organisms have been partially elucidated, and downstream mechanisms in other organisms have been partially clarified. In all studied systems, upon detection of DNA damage in S phase, checkpoint proteins initiate a phosphorylation cascade that ultimately leads to slowing of replication. Upstream signaling in these systems involves the activation of one or more of the phosphatidylinositol-3-kinase-like protein kinases (PIK kinases; ATR and/or ATM in humans, Mec1 and/or Tel1 in budding yeast, and Rad3 in fission yeast). The activated PIK kinases then phosphorylate several proteins, including certain Ser/Thr kinases (Chk1 and/or Chk2 in humans, Rad53 in budding yeast, and Cds1 in fission yeast). These kinases, in turn, phosphorylate other substrates that, directly or indirectly, mediate the slowing of S phase (reviewed in reference 3).In budding yeast, two different mechanisms were shown to slow S phase upon DNA damage by MMS. Of these, one mechanism, inhibition of late-firing origins, depended on the Mec1-Rad53 checkpoint pathway (45, 53), while the other mechanism, inhibition of replication forks, appeared to be a direct consequence of DNA damage rather than a result of checkpoint activation (53). Tercero and Diffley (53) found that, in MMS-treated cells with mutations in the RAD53 gene, unregulated origin firing compensated for checkpoint-independent replication fork slowing, thus permitting a relatively normal overall rate of DNA synthesis. The mechanism by which the Rad53 protein modulates late origin activity is not yet clear, but one possibility is inhibition (by Rad53-catalyzed phosphorylation) of Dbf4, the regulatory subunit of the Cdc7-Dbf4 kinase, which is essential for initiation of replication (7, 8, 14, 55).In vertebrates, at least three different pathways have been shown to contribute to the slowing of S phase after DNA damage. In some cases checkpoint-mediated phosphorylation of Dbf4 inhibits progression through S phase by downregulating origin firing (7, 14), as may take place in budding yeast. In other cases, checkpoint-mediated phosphorylation leads to inhibition and destruction of the protein phosphatase Cdc25A, which is an activator of Cdk2. Cdk2 is the S-phase-specific cyclin-dependent kinase. Cdk2 activity is crucial for initiation of DNA replication and is modulated by inhibitory phosphorylation at Tyr-15. Cdc25A activates Cdk2 by dephosphorylating Tyr-15. Thus, when Cdc25A is phosphorylated by checkpoint kinases after DNA damage and subsequently destroyed, Cdk2 can no longer promote initiation of DNA replication (9, 27). The third mechanism by which vertebrate cells can slow progression through S phase is inhibition of replication fork movement. In vertebrate cells, slowing of replication forks in response to DNA damage is frequently checkpoint dependent; in contrast, in budding yeast, such slowing appeared to be checkpoint independent. In the tested cases, fork slowing has proved to be dependent on the PIK kinase ATR (homologous to budding yeast Mec1 and fission yeast Rad3) and on the Ser/Thr kinase Chk1 (a functional analogue of budding yeast''s Rad53 and fission yeast''s Cds1). In each of these cases, the checkpoint response to DNA damage led to inhibition of origin firing as well as to inhibition of replication fork movement (42, 44, 54). The precise mechanism leading to slowing of replication fork movement has not been fully worked out, but the mechanism appears to involve interactions between Chk1 and the proteins Tim and Tipin (54), whose yeast homologues (Swi1 and Swi3 in fission yeast, Tof1 and Csm3 in budding yeast) form a “replication fork protection complex” that is associated with replication forks (19, 33).Although it is clear that slowing of S phase in response to MMS-induced DNA damage in fission yeast requires both the Rad3 and Cds1 kinases, the pathways operating downstream of Cds1 have been uncertain. We obtained results indicating that Cdc25, which was already known to be a target of Cds1 in hydroxyurea (HU)-treated cells, is also a target of Cds1 in MMS-treated cells, because both overproduction of Cdc25 and conversion of Tyr-15 on Cdc2 (the major cyclin-dependent kinase of fission yeast; also known as Cdk1) to a nonphosphorylatable residue (Cdc2-Y15F; this mutation rendered Cdc2 constitutively active) were sufficient to prevent MMS-induced slowing of S phase (23). We concluded that, in fission yeast, the Rad3→Cds1⊣Cdc25→Cdc2 pathway forms a checkpoint signaling module very similar to the corresponding one of vertebrates. However, Kommajosyula and Rhind were not able to repeat our observations regarding the roles of Cdc25 and Cdc2 (22), so the relevance of Cdc25 and Cdc2 to checkpoint-induced slowing of S phase in fission yeast has remained uncertain until now. In addition, whether S phase in MMS-treated fission yeast cells is slowed by inhibition of origin firing, by reduction in rate of fork movement, or by a combination of these has been equally unclear.In order to resolve these issues, we initiated the series of experiments reported in this paper. To measure the rate of progression through S phase, we followed S phase by flow cytometry and by two-dimensional (2D) gel electrophoresis in cells released from a G₁ block (achieved by incubating cells bearing a cdc10 temperature-sensitive mutation at the restrictive temperature, then releasing to the permissive temperature [21, 23]). We found that, in MMS-treated, checkpoint-competent cells, the firing of early origins near centromeres was partially delayed but that the firing of late origins near telomeres was unaffected. Furthermore, the lifetimes of replication intermediates (RIs) were prolonged, consistent with slowing of replication forks. These effects were completely abrogated both in cells lacking the Cds1 kinase and in cells overproducing the Cdc25 phosphatase, showing that these effects were checkpoint dependent and that the relevant checkpoint pathway probably involved inhibition of Cdc25.     

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.     

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.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

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.     

Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.DNA damage response is an essential cellular guard that protects the genetic material from a constant barrage of genotoxic agents. To ensure their survival after genomic insult, cells orchestrate a signaling cascade that leads to checkpoint-mediated cell cycle arrest and the repair of damaged DNA (16, 35). A failure in this process can have catastrophic cellular consequences leading to the development of numerous disorders such as cancer (18, 30, 32). Because of its intimate connection with human health, deciphering the molecular mechanisms of DNA damage response is of high interest (16, 20).Recently, histone posttranslational modification has emerged as one element that is critical for ensuring a faithful response to genomic challenge (7, 31). An octamer of the four core histones, H3, H4, H2A, and H2B, forms the core protein component of chromatin, and cells possess a considerable number of enzymes that target histones for posttranslation modification (21). These marks can impinge upon many aspects of DNA biology by acting to directly alter chromatin structure or by serving as a binding scaffold for the recruitment of regulatory factors (24).In the context of DNA damage response, one factor that is intimately linked with histone modification is the fission yeast DNA damage checkpoint protein Crb2. After genomic insult, DNA damage checkpoints function to halt cell cycle progression, ensuring sufficient time for lesion repair (16, 35). In the fission yeast Schizosaccharomyces pombe, regulating the transition from G₂ to mitosis (G₂/M) represents the major DNA damage checkpoint and Crb2 is essential for this activity (4, 34). Crb2 is a member of a family of checkpoint regulators that have been termed mediators because they are thought to transmit the checkpoint signal from damage-sensing ATM/ATR-related kinases to effector kinases, such as Chk1, that trigger cell cycle arrest (11, 25). Crb2 is closely related to budding yeast Rad9 and mammalian p53 binding protein 53BP1, which all share two distinct domains, a tandem tudor motif and a pair of C-terminal BRCT repeats (Fig. ​(Fig.1A)1A) (11, 25). Besides 53BP1, Crb2 also shares some functional similarities with other mammalian BRCT-containing checkpoint regulators, such as MDC1 and BRCA1 (11, 25). In response to ionizing irradiation (IR), the rapid accumulation of Crb2 and other checkpoint proteins can be readily visualized as nuclear foci that mark sites of double-strand breaks (DSBs) (9, 25). Understanding the mechanisms that govern this targeting has been an area of intense interest, and for Crb2 this accumulation requires two distinct histone marks: dimethylation of histone H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX) (27, 36).Open in a separate windowFIG. 1.Crb2 pH2AX-binding mutations. (A) Top, schematic representation of Crb2 (not drawn to scale) with relevant mutations indicated. Bottom, protein sequence alignment of a portion of the BRCT phospho-binding motifs from Schizosaccharomyces pombe (sp) Crb2, human (h) 53BP1, human MDC1, and Saccharomyces cerevisiae (sc) Rad9. Identical residues are shaded black; similar residues are shaded gray. *, Crb2 phospho-binding residues. (B) The Crb2 BRCT domains specifically interact with pH2AX. Peptide pulldowns were performed as described in the text with C-terminal fission yeast H2A.1 peptides either unmodified or phosphorylated at Ser129 (see − or + pH2AX) and increasing amounts of the indicated recombinant Crb2 BRCT domain fragments (∼0.1 and 0.3 μM). After binding and washing, SDS-PAGE and Coomassie staining were used to visualize peptide-bound protein. A fraction of the total protein used for binding was also visualized (Input).Mono-, di-, and trimethyl H4K20 are conserved chromatin marks that are readily detectable in fission yeast and mammalian cells (29, 36). In fission yeast, the Kmt5 methylase catalyzes all three H4K20 methyl modifications and its inactivation, or mutation of its H4K20 substrate, severely diminishes Crb2 accumulation at DSBs and compromises checkpoint activity (10, 36). Note that as outlined by the unified nomenclature for the naming of histone lysine methyltransferases (2), the fission yeast H4K20 methylase previously known as Set9 (36) is now termed Kmt5. The requirement for H4K20 methylation is mediated by the tandem tudor domains of Crb2 that preferentially bind H4 tail peptides dimethylated at lysine 20 (3, 14). Tudor motif mutations impair Crb2 DSB targeting and genome integrity in a manner analogous to loss of Kmt5 activity, and dimethylation of H4K20, but not trimethylation, is required for Crb2 activity (10, 14, 42). The tudor domain of 53BP1 can also directly bind H4K20me2, and this recognition event is required for its accumulation at IR-induced DSBs (3, 23, 45).After DNA damage, serine 139 phosphorylation in the mammalian H2A variant H2AX, or a homologous site in canonical yeast H2A, specifically marks sites of genomic lesions (7, 12). The fission yeast genome encodes two H2A proteins, H2A.1 and H2A.2, which differ slightly in their primary amino acid sequence. Phosphorylation of S129 in H2A.1 and S128 in H2A.2 is collectively referred to as phosphorylated H2AX (pH2AX). The ATM/ATR family of PI3-like kinases that includes the fission yeast Rad3 and Tel1 enzymes catalyzes pH2AX (37). H2AX phosphorylation has a critical role in controlling both DNA repair and checkpoint activation in a variety of organisms from yeast to humans (7, 12). Central to its function is the ability of the pH2AX mark to coordinate the recruitment of a number of proteins to genomic lesions, and several factors can directly bind the modification (40). Serine-to-alanine substitutions at the H2AX phosphorylation site in fission yeast H2A (h2ax⁻) severely reduce Crb2 accumulation at IR-induced DSBs and compromise the ability of cells to maintain checkpoint cell cycle arrest in a manner very similar to loss of H4K20 methylation (10, 27).The mechanism underlying the control of Crb2 DSB targeting and checkpoint activation by pH2AX is not understood. Because BRCT domains are known phospho-binding motifs (13), the initial demonstration that pH2AX is required for Crb2 function suggested that direct binding to the modification by Crb2 is critical for checkpoint activity (27). Supporting this idea, it has been demonstrated that the Crb2 BRCT repeats directly and specifically bind pH2AX peptides (22). Structural and biochemical studies have also identified a conserved pH2AX-binding motif in the BRCT repeats of Crb2, budding yeast Rad9, and human MDC1 and 53BP1 (Fig. ​(Fig.1A)1A) (15, 22, 39). As would be expected, mutation of Crb2''s critical phospho-binding motif impairs cell survival after DNA damage (22). Unexpectedly though, loss of its pH2AX-binding activity did not significantly affect the ability of Crb2 to localize to IR-induced DSBs (22). Rather, mutation of the Crb2 pH2AX-binding motif altered the kinetics of Rad22 accumulation at DSBs and triggered a prolonged checkpoint arrest after IR exposure (22). From these observations it was suggested that binding of the Crb2 BRCT repeats to pH2AX is critical for aspects of DNA repair but is not central to Crb2 targeting and checkpoint activity (22).The apparent dispensability of its pH2AX-binding motif in controlling Crb2 localization to IR-induced DSBs (22) was a surprising observation because of the established requirement for the pH2AX modification (10, 27). The extended checkpoint delay seen in Crb2 pH2AX-binding mutants (22) was also unexpected because h2ax⁻ cells cannot maintain checkpoint-mediated cell cycle arrest (10, 27). The prolonged checkpoint arrest was also surprising because a defect in IR-induced Chk1 phosphorylation was observed in the same Crb2 pH2AX-binding mutants (22). For these reasons we sought to reevaluate the requirement for the pH2AX-binding module of Crb2 in controlling DNA damage checkpoint activity. We demonstrate that the critical phospho-coordinating residue of Crb2 is required for binding to pH2AX peptides, Crb2 accumulation at IR-induced DSBs, cell survival after DNA damage, and maintenance of checkpoint-mediated cell cycle arrest. The observed impairments are similar to that reported for abolishment of pH2AX or mutation of the H4K20me2 binding tudor motif of Crb2. Strikingly, a combined ablation of the two modification binding modules of Crb2 produces an additive impairment in checkpoint dysfunction and genome integrity. These results argue that recognition of pH2AX by its BRCT repeats is critical for Crb2 accumulation at genomic lesions and its subsequent checkpoint activity. These observations also corroborate the independent findings of Sofueva et al. (38), who have observed a similar requirement for Crb2 binding to pH2AX in controlling DSB targeting and checkpoint activity.     

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1–3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4–7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR,² is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single- and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were found to play a role in the DNA replication checkpoint (15–17). They interact with the 9-1-1 complex and directly stimulate Mec1/ATR activity in vitro (18–20). Thus, the assembly of multiple protein complexes at stalled DNA replication forks appears to facilitate activation of the DNA replication checkpoint (13, 18).Mrc1 (for mediator of replication checkpoint) was originally identified to be important for cells to respond to hydroxyurea in S. cerevisiae and Schizosaccharomyces pombe (21, 22). Mrc1 is a component of the DNA replisome and travels with the replication forks along chromosome during DNA synthesis (23–25). Deletion of MRC1 causes defects in DNA replication, indicating its role in the normal progression of DNA replication (23). Interestingly, when DNA replication is blocked by hydroxyurea, Mrc1 undergoes Mec1- and Rad3 (S. pombe ortholog of Mec1)-dependent phosphorylation (21, 22). In S. cerevisiae, mutations of Mrc1 at the (S/T)Q sites, which are consensus phosphorylation sites of the Mec1/ATR family kinases, abolishes hydroxyurea-induced Mrc1 phosphorylation in vivo, suggesting a direct phosphorylation of Mrc1 by Mec1 (21, 22).Rad53 and Cds1, homologs of human Chk2, are the major effector kinases in the DNA replication checkpoints in S. cerevisiae and S. pombe, respectively. Activation of Rad53 is a hallmark of DNA replication checkpoint activation and is important for the maintenance of DNA replication forks in response to DNA replication stress (5, 6). Thus, it is important to understand how Rad53 activity is controlled. Interestingly, mutation of all the (S/T)Q sites of Mrc1 not only abolishes the phosphorylation of Mrc1 by Mec1 but also compromises hydroxyurea-induced Rad53 activation in S. cerevisiae (21). Similarly, mutation of the TQ sites of Mrc1 in S. pombe was shown to abolish the binding between Cds1 and Mrc1 as well as Cds1 activation (22). Further, mutation of specific TQ sites of Mrc1 in S. pombe abolishes its binding to Cds1 in vitro and the activation of Cds1 in vivo (26). Thus, Mec1/Rad3-dependent phosphorylation of Mrc1 is responsible for Mrc1 binding to Rad53/Cds1, which is essential for Rad53/Cds1 activation.An intriguing property of the Chk2 family kinases is their ability to undergo autophosphorylation and activation in the absence of other proteins in vitro (27, 28). First, autophosphorylation of a conserved threonine residue in the activation loop of Chk2 family kinase was found to be an essential part of their activation processes (26, 29–31). Second, a direct and trans-phosphorylation of the N-terminal TQ sites of the Chk2 family kinases by the Mec1/ATR family kinases is also important for their activation in vivo. Analogous to the requirement of N-terminal TQ site phosphorylation of Chk2 by ATR in human (32), the activation of Rad53/Cds1 in vivo requires phosphorylation of TQ sites in their N termini by Mec1/Rad3 (33, 34).Considering that Mec1, Mrc1, and many other proteins are recruited at stalled DNA replication forks and have been shown to be involved in DNA replication checkpoint activation, a key question remains unresolved: what is the minimal system that is capable of activating Rad53 directly? Given the direct physical interaction between Mrc1 and Rad53 and the requirement of Mrc1 and Mec1 in vivo, it is likely that they both play a role in Rad53 activation. Furthermore, what is the molecular mechanism of Rad53 activation by its upstream activators? To address these questions, a faithful reconstitution of the activation of Rad53 using purified proteins is necessary. In this study, we developed an activity-based assay consisting of the Dun1 kinase, a downstream substrate of Rad53, and Sml1, as a substrate of Dun1, to quantitatively measure the activity of Rad53. Using this coupled kinase assay from Rad53 to Dun1 and then to Sml1, we screened for Mrc1 and its associated factors to see whether they could directly activate Rad53 in vitro. Our results showed that Mec1 and Mrc1 collaborate to constitute a minimal system in direct activation of Rad53.