CDK phosphorylation of Drc1 regulates DNA replication in fission yeast

Cyclin-dependent kinases (CDKs) are absolutely required for DNA replication in eukaryotic cells. CDKs are thought to activate one or more replication factors, but the identities of these proteins are unknown. Here we describe fission yeast Drc1, a protein required for DNA replication that is phosphorylated by Cdc2. Drc1 depletion leads to catastrophic mitotic divisions with incompletely replicated DNA, indicating that Drc1 is required for DNA synthesis and S-M replication checkpoint control. Drc1 associates with Cdc2 and is phosphorylated at the onset of S phase when Cdc2 is activated. Mutant Drc1 that lacks CDK phosphorylation sites is nonfunctional and fails to interact with Cut5 replication factor. These data suggest that Cdc2 promotes DNA replication by phosphorylating Drc1 and regulating its association with Cut5.     

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.     

A novel intermediate in initiation complex assembly for fission yeast DNA replication

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.     

DNA replication: mammalian Treslin-TopBP1 interaction mirrors yeast Sld3-Dpb11

There are many parallels between DNA replication in yeast and humans. Now,?two recent studies extend this relationship by dissecting key conserved interactions necessary for initiation of the replisome.     

Regulatory mechanism of the initiation step of DNA replication by CDK in budding yeast

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation.     

Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.     

Treslin, DUE-B, and GEMC1 cannot complement Sld3 mutants in fission yeast

Initiation of DNA replication in eukaryotes is an evolutionarily conserved process that involves two distinct steps: the formation of prereplication complexes at replication origins in G1 and the assembly of preinitiation complexes (pre-ICs) in S phase, which leads to activation of the replication helicase. For the assembly of pre-ICs in yeast, formation of the Sld2-Dpb11-Sld3 complex is a critical event that requires phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. In mammals, RecQL4 and TopBP1 are excellent ortholog candidates for Sld2 and Dpb11, respectively. In this past year, three TopBP1-interacting proteins Treslin/Ticrr, GEMC1, and DUE-B have been identified in metazoans as possible functional orthologs of the yeast Sld3. To test this hypothesis, we carried out several complementation tests in fission yeast. The proteins were expressed at various levels in the temperature-sensitive sld3-10 mutant and in cells that lack endogenous Sld3. Our result showed that none of these metazoan proteins could rescue growth defect of the sld3 mutants. Although the result may have several interpretations, it is possible that the helicase activation in mammals has diverged in complexity during evolution from that in yeasts and may involve multiple players that interact with TopBP1.     

Regulation of DNA replication machinery by Mrc1 in fission yeast

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.     

Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication

Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S-phase cyclin-dependent kinase (S-CDK)-independent binding is sufficient for DNA replication while S-CDK-dependent binding is dispensable. Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.     

DNA replication origins fire stochastically in fission yeast

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.     

Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast

A checkpoint responding to DNA damage in G2 results in a delay in the onset of mitosis through inhibition of p34^cdc2 kinase activity via maintenance of inhibitory tyrosine phosphorylation. Genetic analyses of this checkpoint in fission yeast have identified single alleles of several genes, suggesting these screens are not yet saturating, and hence further genes await identification. To fully understand the complexity of this checkpoint it will be necessary to define all the genes involved. To this end we screened for new mutants defective in the ability to delay mitosis in the presence of DNA-damaging agents. Twenty-four mutants were isolated that were defective in UV-C and MMS-induced checkpoint delay. Amongst these mutants was an allele of cut5 that was also defective in the checkpoint responses. We show here, contrary to previous reports, that the UV-C induced checkpoint response is defective in cut5 mutants. Therefore, like all other checkpoint mutants, cut5 is required for G2 checkpoint arrest following DNA damage, regardless of the nature of the lesions involved. Received: 24 July 1998 / Accepted: 14 September 1998     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine

We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 μM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.     

Genome-wide characterization of fission yeast DNA replication origins

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.     

Regulation of DNA replication through Sld3-Dpb11 interaction is conserved from yeast to humans

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.     

Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.     

Checkpoint independence of most DNA replication origins in fission yeast

Background

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results

Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion

The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.     

Complex mechanism of site-specific DNA replication termination in fission yeast

A site-specific replication terminator, RTS1, is present at the Schizosaccharomyces pombe mating-type locus mat1. RTS1 regulates the direction of replication at mat1, optimizing mating-type switching that occurs as a replication-coupled recombination event. Here we show that RTS1 contains two cis-acting sequences that cooperate for efficient replication termination. First, a sequence of approximately 450 bp containing four repeated 55 bp motifs is essential for function. Secondly, a purine-rich sequence of approximately 60 bp without intrinsic activity, located proximal to the repeats, acts cooperatively to increase barrier activity 4-fold. Our data suggest that the trans-acting factors rtf1p and rtf2p act through the repeated motifs and the purine-rich element, respectively. Thus, efficient site-specific replication termination at RTS1 occurs by a complex mechanism involving several cis-acting sequences and trans-acting factors. Interestingly, RTS1 displays similarities to mammalian rDNA replication barriers.     

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD⁺-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.