Biomedical subjects

Katsunori Sugimoto

Publications and source records attributed to Katsunori Sugimoto.

10 recordsLinked to original sources

PubMed2006

ATR homolog Mec1 controls association of DNA polymerase zeta-Rev1 complex with regions near a double-strand break.

DNA polymerase zeta (Polzeta) and Rev1 contribute to the bypassing of DNA lesions, termed translesion DNA synthesis (TLS). Polzeta consists of two subunits, one encoded by REV3 (the catalytic subunit) and the other encoded by REV7. Rev1 acts as a deoxycytidyl transferase, inserting dCMP opposite lesions. Polzeta and Rev1 have been shown to operate in the same TLS pathway in the budding yeast Saccharomyces cerevisiae. Here, we show that budding yeast Polzeta and Rev1 form a complex and associate together with double-strand breaks (DSBs). As a component of the Polzeta-Rev1 complex, Rev1 plays a noncatalytic role in the association with DSBs. In budding yeast, the ATR-homolog Mec1 plays a central role in the DNA-damage checkpoint response. We further show that Mec1-dependent phosphorylation promotes the Polzeta-Rev1 association with DSBs. Rev1 association with DSBs requires neither the function of the Rad24 checkpoint-clamp loader nor the Rad6-Rad18-mediated ubiquitination of PCNA. Our results reveal a novel role of Mec1 in the localization of the Polzeta-Rev1 complex to DNA lesions and highlight a linkage of TLS polymerases to the checkpoint response.

DNA Damage↗

PubMed2005

Role of the C terminus of Mec1 checkpoint kinase in its localization to sites of DNA damage.

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), coordinate the cellular response to DNA damage. In budding yeast, ATR homologue Mec1 plays a central role in DNA damage signaling. Mec1 interacts physically with Ddc2 and functions in the form of the Mec1-Ddc2 complex. To identify proteins interacting with the Mec1-Ddc2 complex, we performed a modified two-hybrid screen and isolated RFA1 and RFA2, genes that encode subunits of replication protein A (RPA). Using the two-hybrid system, we found that the extreme C-terminal region of Mec1 is critical for RPA binding. The C-terminal substitution mutation does not affect the Mec1-Ddc2 complex formation, but it does impair the interaction of Mec1 and Ddc2 with RPA as well as their association with DNA lesions. The C-terminal mutation also decreases Mec1 kinase activity. However, the Mec1 kinase-defect by itself does not perturb Mec1 association with sites of DNA damage. We also found that Mec1 and Ddc2 associate with sites of DNA damage in an interdependent manner. Our findings support the model in which Mec1 and Ddc2 localize to sites of DNA damage by interacting with RPA in the form of the Mec1-Ddc2 complex.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The reconstituted human Chl12-RFC complex functions as a second PCNA loader.

The sister chromatid cohesion factor Chl12 shares amino acid sequence similarity with RFC1, the largest subunit of replication factor C (RFC), and forms a clamp loader complex in association with the RFC small subunits RFCs2-5. It has been shown that the human Chl12-RFC complex, reconstituted with a baculovirus expression system, specifically interacts with human proliferating cell nuclear antigen (PCNA). The purified Chl12-RFC complex is structurally indistinguishable from RFC, as shown by electron microscopy, and it exhibits DNA-stimulated ATPase activity that is further enhanced by PCNA, and by DNA binding activity on specific primer/template DNA structures. Furthermore, the complex loads PCNA onto a circular DNA substrate, and stimulates DNA polymerase delta DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2004

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Cell Cycle Proteins↗

PubMed2004

Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism.

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.

Amino Acid Motifs↗

PubMed2003

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex.

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Bromodeoxyuridine↗

PubMed2003

ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism.

In budding yeast, TEL1 encodes a protein closely related to ATM. Xrs2 is an Nbs1 homolog and forms a complex with Mre11 and Rad50. We show here that Tel1 associates with double-strand breaks (DSBs) through a mechanism dependent on the C terminus of Xrs2. Although Xrs2 is required for the DNA degradation at DSBs, the C-terminal Xrs2 truncation does not affect the degradation. Tel1 and the C terminus of Xrs2 are similarly involved in cell survival and Rad53 phosphorylation after DNA damage. Our findings suggest that the Tel1 association with DNA lesions is required for the activation of DNA damage responses.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2003

The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment.

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.

Cell Cycle↗

PubMed2002

A proteomics approach to identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates. Identification of the human CHL12/RFCs2-5 complex as a novel PCNA-binding protein.

Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase delta but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding proteins by a combination of affinity chromatography and mass spectrometric analyses. We detected more than 20 specific protein bands of various intensities in fractions bound to PCNA-fixed resin from human cell lysates and determined their peptide sequences by liquid chromatography and tandem mass spectrometry. A search with human protein data bases identified 12 reported PCNA-binding proteins from both cytoplasmic (S100 lysate) and nuclear extracts with substantial significance and four more solely from the nuclear preparation. CHL12, a factor involved in checkpoint response and chromosome cohesion, was a novel example found in both lysates. Further studies with recombinant proteins demonstrated that CHL12 and small subunits of replication factor C form a complex that interacts with PCNA.

Base Sequence↗

PubMed2002

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.

Base Sequence↗