Chiaki Noguchi

Swi1 and Swi3 Are Components of a Replication Fork Protection Complex in Fission Yeast

ABSTRACT Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3+. The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Δ and swi3Δ mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Δ or swi3Δ cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.

ABSTRACT The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion.

The Timeless-Tipin protein complex has been reported to be important for replication checkpoint and normal DNA replication processes. However, the precise mechanisms by which Timeless-Tipin preserves genomic integrity are largely unclear. Here, we describe the roles of Timeless-Tipin in replication fork stabilization and sister chromatid cohesion. We show in human cells that Timeless is recruited to replication origin regions and dissociate from them as replication proceeds. Cdc45, which is known to be required for replication fork progression, shows similar patterns of origin association to those of Timeless. Depletion of Timeless-Tipin causes chromosome fragmentation and defects in damage repair in response to fork collapse, suggesting that it is required for replication fork maintenance under stress. We also demonstrate that depletion of Timeless-Tipin impairs sister chromatid cohesion and causes a defect in mitotic progression. Consistently, Timeless-Tipin co-purifies with cohesin subunits and is required for their stable association with chromatin during S phase. Timeless associates with the cohesion-promoting DNA helicase ChlR1, which, when overexpressed, partially alleviates the cohesion defect of cells depleted of Timeless-Tipin. These results suggest that Timeless-Tipin functions as a replication fork stabilizer that couples DNA replication with sister chromatid cohesion established at replication forks.

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.

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.

Sap1 Promotes the Association of the Replication Fork Protection Complex with Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces Pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.

Coordinated Degradation of Replisome Components Ensures Genome Stability upon Replication Stress in the Absence of the Replication Fork Protection Complex

The stabilization of the replisome complex is essential in order to achieve highly processive DNA replication and preserve genomic integrity. Conversely, it would also be advantageous for the cell to abrogate replisome functions to prevent inappropriate replication when fork progression is adversely perturbed. However, such mechanisms remain elusive. Here we report that replicative DNA polymerases and helicases, the major components of the replisome, are degraded in concert in the absence of Swi1, a subunit of the replication fork protection complex. In sharp contrast, ORC and PCNA, which are also required for DNA replication, were stably maintained. We demonstrate that this degradation of DNA polymerases and helicases is dependent on the ubiquitin-proteasome system, in which the SCFPof3 ubiquitin ligase is involved. Consistently, we show that Pof3 interacts with DNA polymerase ε. Remarkably, forced accumulation of replisome components leads to abnormal DNA replication and mitotic catastrophes in the absence of Swi1. Swi1 is known to prevent fork collapse at natural replication block sites throughout the genome. Therefore, our results suggest that the cell elicits a program to degrade replisomes upon replication stress in the absence of Swi1. We also suggest that this program prevents inappropriate duplication of the genome, which in turn contributes to the preservation of genomic integrity.

A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a popular model organism to study various cellular processes, although research tools available for S. pombe are relatively inadequate. To facilitate genetic and biochemical investigation in S. pombe, we report here a system of vectors for genomic FLAG epitope‐tagging. These vectors enable us to amplify gene‐targeting fragments for integration into specific loci of the S. pombe genome. All vectors in this report were designed to express FLAG epitope‐tagged proteins from their endogenous genomic loci. Vectors for N‐terminal FLAG epitope‐tagging allow us to control protein expression levels using the wild‐type nmt1 promoter, its weaker derivatives, and the urg1 promoter. These vectors are available with various antibiotic markers including kanMX6, hphMX6, natMX6 and bleMX6, and the his3+ marker. Vectors for C‐terminal FLAG epitope‐tagging were designed to express FLAG‐fusion proteins under the control of their native promoters at their own genomic loci, allowing us to characterize protein functions under physiological conditions. These vectors are available with kanMX6, hphMX6, nat‐MX6 and bleMX6 markers. The series of vectors described in this report should prove useful for protein studies in fission yeast.

Assays Used to Study the DNA Replication Checkpoint in Fission Yeast

The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components and preserving DNA structures. Here we describe a series of assays used to study the replication checkpoint. These assays allow us to investigate the specific functions of proteins involved in the replication checkpoint in fission yeast.

The Double-Bromodomain Proteins Bdf1 and Bdf2 Modulate Chromatin Structure to Regulate S-Phase Stress Response in Schizosaccharomyces pombe

Bromodomain proteins bind acetylated histones to regulate transcription. Emerging evidence suggests that histone acetylation plays an important role in DNA replication and repair, although its precise mechanisms are not well understood. Here we report studies of two double bromodomain-containing proteins, Bdf1 and Bdf2, in fission yeast. Loss of Bdf1 or Bdf2 led to a reduction in the level of histone H4 acetylation. Both bdf1Δ and bdf2Δ cells showed sensitivity to DNA damaging agents, including camptothecin, that cause replication fork breakage. Consistently, Bdf1 and Bdf2 were important for recovery of broken replication forks and suppression of DNA damage. Surprisingly, deletion of bdf1 or bdf2 partially suppressed sensitivity of various checkpoint mutants including swi1Δ, mrc1Δ, cds1Δ, crb2Δ, chk1Δ, and rad3Δ, to hydroxyurea, a compound that stalls replication forks and activates the Cds1-dependent S-phase checkpoint. This suppression was not due to reactivation of Cds1. Instead, we found that bdf2 deletion alleviates DNA damage accumulation caused by defects in the DNA replication checkpoint. We also show that hydroxyurea sensitivity of mrc1Δ and swi1Δ was suppressed by mutations in histone H4 acetyltransferase subunits or histone H4. These results suggest that the double bromodomain-containing proteins modulate chromatin structure to coordinate DNA replication and S-phase stress response.