Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.     

Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.     

FHA domain-mediated DNA checkpoint regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.     

Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.     

Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control

BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.     

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double‐strand breaks (DSBs), but only in the G1‐phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1–2 and 7–8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1–2 and 4–5. The BRCT domains 4–5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S‐phase, we observed a checkpoint defect after siRNA‐mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.     

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.     

Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.     

Histone H3 K56 acetylation, chromatin assembly, and the DNA damage checkpoint

The role of chromatin and its modulation during DNA repair has become increasingly understood in recent years. A number of histone modifications that contribute towards the cellular response to DNA damage have been identified, including the acetylation of histone H3 at lysine 56. H3 K56 acetylation occurs normally during S phase, but persists in the presence of DNA damage. In the absence of this modification, cellular survival following DNA damage is impaired. Two recent reports provide additional insights into how H3 K56 acetylation functions in DNA damage responses. In particular, this modification appears to be important for both normal replication-coupled nucleosome assembly as well as nucleosome assembly at sites of DNA damage following repair.     

The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange

Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro. To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination. In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids. Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences. This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases. In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis. Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.     

Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases

First described in Arabidopsis thaliana, Tousled-like kinases (Tlks) are highly conserved in both plants and animals. In plants, Tousled kinase is essential for proper flower and leaf development, but no direct functional link to any other plant gene product has yet been established. Likewise, the role of Tlks in animals is unknown. In human cells, two structurally similar Tlks, Tlk1 and Tlk2, were recently shown to be cell cycle-regulated kinases with maximal activities during S phase. Here, we report the identification of two human homologs of the Drosophila chromatin assembly factor Asf1 (anti-silencing function 1) as physiological substrates of Tlks. We show that human Asf1 proteins are phosphorylated by Tlks both in vivo and in vitro. Furthermore, Asf1 proteins are phosphorylated during S phase, when Tlks are maximally active. Conversely, Asf1 proteins are dephosphorylated upon the activation of the DNA replication checkpoint, concomitant with the rapid inactivation of Tlks. These data indicate that Tlk family members regulate chromatin assembly during DNA replication, and they suggest a plausible explanation for the pleiotropic developmental defects of plant tousled mutants.     

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.     

Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p

Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor. Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution. In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease. We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo.     

Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage.

Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.