The DNA helicase activity of yeast Sgs1p is essential for normal lifespan but not for resistance to topoisomerase inhibitors

The Saccharomyces cerevisiae SGS1 gene is a member of the RecQ family of ATP-dependent DNA helicases, which includes the human WRN, BLM and RECQ4 genes. Mutations in the WRN gene cause the human premature ageing disorder, Werner's syndrome. Deletion of the SGS1 gene also causes premature ageing in yeast, suggesting that the molecular mechanisms of cellular ageing may be evolutionarily conserved. To investigate the role of the RecQ helicase domain in ageing, a point mutation (SGS1 K(706)-->A) known to eliminate the DNA helicase activity of Sgs1p was constructed. This mutant allele failed to rescue the premature ageing of the sgs1Delta strain, demonstrating that Sgs1p DNA helicase activity is required for a normal lifespan. In contrast, the SGS1 K(706)-->A allele was sufficient to rescue the hypersensitivity of the sgs1Delta strain to topoisomerase inhibitors, but not other genotoxic agents. These findings support the idea that Sgs1p fulfils multiple cellular functions, and that DNA helicase activity is dispensable for some of these (e.g. functional interaction with topoisomerases), but essential for others (e.g. longevity).     

The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes

The Saccharomyces cerevisiae DNA helicase Rrm3p is needed for normal fork progression through >1000 discrete sites scattered throughout the genome. Here we show that replication of all yeast chromosomes was markedly delayed in rrm3 cells. Delayed replication was seen even in a region that lacks any predicted Rrm3p-dependent sites. Based on the pattern of replication intermediates in two-dimensional gels, the rate of fork movement in rrm3 cells appeared similar to wild-type except at known Rrm3p-dependent sites. These data suggest that although Rrm3p has a global role in DNA replication, its activity is needed only or primarily at specific, difficult-to-replicate sites. By the criterion of chromatin immunoprecipitation, Rrm3p was associated with both Rrm3p-dependent and -independent sites, and moved with the replication fork through both. In addition, Rrm3p interacted with Pol2p, the catalytic subunit of DNA polymerase epsilon, in vivo. Thus, rather than being recruited to its sites of action when replication forks stall at these sites, Rrm3p is likely a component of the replication fork apparatus.     

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase

S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. We have studied the RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template in sgs1 cells. We show that, in sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein, is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. We suggest that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. Our findings might contribute to explaining some of the recombination defects of BLM cells.     

Importance of the Sgs1 helicase activity in DNA repair of Saccharomyces cerevisiae

 The Saccharomyces cerevisiae Sgs1 protein, together with Schizosaccharomyces pombe Rqh1 and the human Bloom and Werner proteins, is a DNA helicase of the Escherichia coli RecQ family. Mutation of SGS1 causes premature aging in yeast cells, including the accumulation of extrachromosomal rDNA circles. We have recently shown that Sgs1p interacts with the DNA repair Rad16p protein and is epistatic to Rad16p for UVC, 4-NQO and H₂O₂ lesions. Therefore we tested sgs1 strains containing mutations in the helicase and C-terminal domains. We demonstrate here that the helicase activity of the Sgs1 is important for most elements of the sgs1 mutation phenotype, including sensitivity to UVC, 4-NQO, H₂O₂, MMS and hydroxyurea. Received: 8 August / 18 October 1999     

Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast

The DNA replication checkpoint is a complex signal transduction pathway, present in all eukaryotic cells, that functions to maintain genomic integrity and cell viability when DNA replication is perturbed. In Schizosaccharomyces pombe the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. We report that the replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites in Mrc1. Cds1 is then primed for activation by Rad3-dependent phosphorylation. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation. This two-stage activation mechanism for the replication checkpoint allows for rapid activation with a high signal-to-noise ratio.     

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51

BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.     

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.     

Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53

When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication fork. Blocking initiation of DNA replication blocks Mrc1 loading onto origins, providing an explanation for why so many mutants in DNA replication show checkpoint defects. In the presence of replication blocks, we find that Mec1 is recruited to regions of stalled replication, where it encounters and presumably phosphorylates Mrc1. Mutation of the canonical Mec1 phosphorylation sites on Mrc1 prevents Mrc1 phosphorylation and blocks Rad53 activation, but does not alter Mrc1's role in DNA replication. Our results suggest a model whereby in response to DNA replication interference, the Mec1 kinase is recruited to sites of replication blocks and phosphorylates a component of the DNA replication complex, Mrc1, thereby setting up a solid-state Rad53 activation platform to initiate the checkpoint response.     

Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction.

We have previously shown that the pharmacological agents 4-(2-aminoethyl)=benzenesulfonylfluoride hydrochloride (AEBSF) and Na-p-tosyl-L-lysine chloromethylketone (TLCK), inhibitors of trypsin-like serine proteases, prevent the death of trophic factor-deprived PC12 cells and sympathetic neurons. Both AEBSF and TLCK inhibit caspase activation in this model, but it is unclear whether they do so indirectly or through a direct effect at the level of the caspases. In the current study, we have used these agents in another model of neuronal death that is induced by DNA damage. We find that both agents delay the death of DNA-damaged PC12 cells, neonatal rat sympathetic neurons and embryonic rat cortical neurons. As in the trophic deprivation model, they act upstream of the caspases. In addition, they prevent mitochondrial alterations, such as cytochrome c release or loss of transmembrane potential. In contrast, the general caspase inhibitor bok-asp-fmk does not prevent cytochrome c release and has only a partial and transient effect on loss of transmembrane potential. Interestingly, both AEBSF and TLCK prevent the induction and nuclear accumulation of p53 that is induced by DNA damage in cortical neurons. Therefore, these serine protease inhibitors act at a point upstream in the apoptotic pathway, prior to p53 induction and the mitochondrial checkpoint, to delay neuronal death in this model, and do not act at the level of the caspases.We conclude that therapeutic strategies based on serine protease inhibition may be useful in preventing neuronal cell death.     

Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast

Unrepaired DNA lesions in the template strand block the replication fork. In yeast, Mec1 protein kinase-mediated replication checkpoint prevents the breakdown of replication forks and maintains viability in DNA-damaged cells going through the S phase. By ensuring that the replisome does not dissociate from the fork stalled at the lesion site, the replication checkpoint presumably coordinates the action of lesion bypass processes with the replisome. However, it has remained unclear as to which of the lesion bypass processes—translesion synthesis (TLS) and/or template switching—depend on the activation of the replication checkpoint. Here we determine if the Mec1 kinase and the subunits of the checkpoint clamp and the clamp loader are required for TLS. We show that proficient TLS can occur in the absence of these checkpoint proteins in nucleotide excision repair (NER)-proficient cells; however, in the absence of NER, checkpoint protein-mediated Rev1 phosphorylation contributes to increasing the proficiency of DNA polymerase ζ-dependent TLS.     

A mutation in yeast Tel1p that causes differential effects on the DNA damage checkpoint and telomere maintenance

ATM/ATR homologs are the central elements of genome surveillance mechanisms in many organisms, including yeasts, flies, and mammals. In Saccharomyces cerevisiae, most checkpoint responses depend on the ATR ortholog Mec1p. The yeast ATM ortholog, Tel1p, so far has been implicated in a specific DNA damage checkpoint during S-phase as well as in telomere homeostasis. In particular, yeast cells lacking only Tel1p harbor short but stable telomeres, while cells lacking both Tel1p and Mec1p are unable to maintain telomeric repeats and senesce. Here, we present the characterization of a new mutation in the TEL1-gene, called tel1-11, which was isolated by virtue of a synthetic lethal interaction at 37°C with a previously described mec1-ts mutation. Interestingly, telomere and checkpoint functions are differentially affected by the mutant protein Tel1-11p. The Tel1p-dependent checkpoint response is undetectable in cells containing Tel1-11p and incubated at 37°C, but basic telomere function is maintained. Further, when the same cells are incubated at 26°C, Tel1-11p confers full proficiency for all telomere functions analyzed, whereas the function for DNA-damage checkpoint activation is clearly affected. The results thus strongly suggest that the different cellular pathways affected by Tel1p do not require the same level of Tel1p activity to be fully functional. Electronic Supplementary Material Supplementary material is available for this article at .     

The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.     

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication

The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.     

Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53

Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G(1) arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1).     

Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint

Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.     

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.     

Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast

We report here that a normal budding yeast chromosome (ChrVII) can undergo remarkable cycles of chromosome instability. The events associated with cycles of instability caused a distinctive "sectoring" of colonies on selective agar plates. We found that instability initiated at any of several sites on ChrVII, and was sharply increased by the disruption of DNA replication or by defects in checkpoint controls. We studied in detail the cycles of instability associated with one particular chromosomal site (the "403 site"). This site contained multiple tRNA genes known to stall replication forks, and when deleted, the overall frequency of sectoring was reduced. Instability of the 403 site involved multiple nonallelic recombination events that led to the formation of a monocentric translocation. This translocation remained unstable, frequently undergoing either loss or recombination events linked to the translocation junction. These results suggest a model in which instability initiates at specific chromosomal sites that stall replication forks. Forks not stabilized by checkpoint proteins break and undergo multiple rounds of nonallelic recombination to form translocations. Some translocations remain unstable because they join two "incompatible" chromosomal regions. Cycles of instability of this normal yeast chromosome may be relevant to chromosome instability of mammalian fragile sites and of chromosomes in cancer cells.     

MRC1-dependent scaling of the budding yeast DNA replication timing program

We describe the DNA replication timing programs of 14 yeast mutants with an extended S phase identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control, and dNTP synthesis and affect different parts of S phase. In 13 of the mutants, origin activation time scales with the duration of S phase. A limited number of origins become inactive in these strains, with inactive origins characterized by small replicons and distributed throughout S phase. In sharp contrast, cells deleted of MRC1, a gene implicated in replication fork stabilization and in the replication checkpoint pathway, maintained wild-type firing times despite over twofold lengthening of S phase. Numerous dormant origins were activated in this mutant. Our data suggest that most perturbations that lengthen S phase affect the entire program of replication timing, rather than a specific subset of origins, maintaining the relative order of origin firing time and delaying firing with relative proportions. Mrc1 emerges as a regulator of this robustness of the replication program.Eukaryotic cells replicate their DNA in a highly orchestrated manner. DNA synthesis is initiated at specific chromosomal sites (replication origins) and proceeds in a bidirectional manner. Different loci are replicated at different times during S phase, depending on their distance from the nearest replication origin and on the time at which that origin initiates replication. This temporal order is highly reproducible between cells and can change concomitantly with cell differentiation (Hiratani et al. 2008; Schwaiger et al. 2009).The process of DNA replication has been studied extensively. Replication origins are licensed during mitosis and early G₁, with the binding of ORC (origin recognition complex) and the subsequent recruitment of the minichromosome maintenance (MCM) complex (part of the replicative DNA helicase) (for review, see Bell and Dutta 2002; Aladjem 2007; Sclafani and Holzen 2007). The onset of S phase is marked by the induction of the Clb5-6 cyclins (for review, see Murray 2004) and the associated degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1 (Verma et al. 1997), leading to the phosphorylation of MCM subunits (via the cell cycle kinase Cdc7–Dbf4), and the recruitment of DNA polymerases α and ɛ to licensed origins (via phosphorylated Sld2/Sld3; Tanaka et al. 2007; Zegerman and Diffley 2007). Origins are then activated, with DNA polymerases catalyzing the addition of dNTPs to primer–template junctions.Only little is known about the mechanism that regulates the timing at which specific origins are activated. Saccharomyces cerevisiae sic1 mutants, which enter S phase prematurely, replicate DNA from a smaller number of origins, presumably because of a failure to license a subset of origins (Lengronne and Schwob 2002). Late replication origins are inactive when the S phase cyclin CLB5 is deleted (Donaldson et al. 1998) and when cells are subjected to DNA damage (by methyl methanesulfonate [MMS]) or treated with hydroxyurea (HU), a drug that reversibly inhibits ribonucleotide reductase (RNR) required for reduction of nucleoside triphosphates (NTPs) to dNTPs. Notably, the replication checkpoint kinases Mec1 and Rad53 are required for this suppression (Santocanale and Diffley 1998; Shirahige et al. 1998). Recent work, however, demonstrated that rather than suppressing late origins, HU confers an overall slowdown of the replication program (Alvino et al. 2007).To obtain further insight into the regulators of the replication program, we performed a genome-wide screen for mutants with an extended S phase. A total of 14 genes were identified, nine of which were not previously associated with S phase duration. In 13 of the mutants, origin firing time appeared to scale with the duration of S phase. Scaling was lost, and numerous dormant origins were activated, in cells deleted for MRC1, a gene required for normal fork progression (Szyjka et al. 2005; Tourriere et al. 2005) and for transducing the checkpoint signal from Mec1 to Rad53 upon replication stress (Osborn and Elledge 2003).     

S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe

Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest. Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis. Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents. Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G₁ or G₂. Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26. Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response. We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins. This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins.     

Clamp and clamp loader structures of the human checkpoint protein complexes,Rad9-1-1 and Rad17-RFC

BACKGROUND: We have reported that protein imaging by transmission electron microscope observation based on low-angle platinum shadowing can reproduce characteristic ring structures of the replication clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader protein, replication factor C (RFC). The checkpoint protein complexes, Rad9-Hus1-Rad1 (Rad9-1-1) and Rad17-RFCs2-5 (Rad17-RFC), have been predicted to function as novel clamp and clamp loader proteins, respectively, due to their amino acid sequence similarities with PCNA and RFC. RESULTS: We reconstituted human Rad9-1-1 and Rad17-RFC complexes in insect cells using a baculovirus expression system and showed purified Rad9-1-1 to be composed of equimolar amounts of Rad9, Hus1 and Rad1 proteins, exhibiting a native molecular mass of 100 kDa, in line with a trimeric complex. When Rad17 was co-expressed with the four small subunits of RFC in insect cells, these proteins formed a complex of 240 kDa that displayed DNA binding, ATPase activity and binding to its predicted target protein, Rad9-1-1. Analyses of the molecular architecture of Rad9-1-1 and Rad17-RFC using transmission electron microscopy, in comparison with PCNA and RFC, revealed the Rad9-1-1 complex to have a characteristic ring structure indistinguishable from that of PCNA in shape and size. In addition, the Rad17-RFC complex was found to be oval in structure and 26 x 22 nm in size with a cleft, reminiscent of the structure of RFC. CONCLUSION: Our direct comparison of images from the two sets of clamp and clamp loader proteins indicated that Rad9-1-1 and Rad17-RFC are, respectively, structural orthologs of PCNA and RFC, with presumed functions as novel clamp and clamp-loader proteins in eukaryotes.