Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

ABSTRACT Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase ɛ (Dpb11-Polɛ complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polɛ associated with these fragments. Association of Dpb11 and Polɛ with these fragments was mutually dependent, suggesting that the Dpb11-Polɛ complex associates with the ARS. Moreover, Dpb11 was required for the association of Polα-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polɛ complex with the ARS fragments is required for the association of the Polα-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polɛ in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases α and ɛ and the ARS.

[1]  H. Wang,et al.  DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  W. L. Fangman,et al.  A yeast origin of replication is activated late in S phase , 1991, Cell.

[3]  M. Bjornsti,et al.  CDC45 and DPB11 are required for processive DNA replication and resistance to DNA topoisomerase I-mediated DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  上村 陽一郎.  Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication , 1998 .

[5]  G. Lucchini,et al.  Mutations in the gene encoding the 34 kDa subunit of yeast replication protein A cause defective S phase progression. , 1995, Journal of molecular biology.

[6]  M. O'Connell,et al.  Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast , 1998, Molecular and General Genetics MGG.

[7]  K. Luo,et al.  SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. , 1997, Genes & development.

[8]  B. Tye.  The MCM2-3-5 proteins: are they replication licensing factors? , 1994, Trends in cell biology.

[9]  W. L. Fangman,et al.  A question of time: Replication origins of eukaryotic chromosomes , 1992, Cell.

[10]  L. Drury,et al.  Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Yanagida,et al.  Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. , 1997, Genes & development.

[12]  A. Riggs,et al.  On the mechanism of DNA replication in mammalian chromosomes. , 1968, Journal of molecular biology.

[13]  M. Lei,et al.  Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[14]  K Nasmyth,et al.  Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin‐ and Dbf4‐dependent kinases , 1998, The EMBO journal.

[15]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[16]  O. Aparicio,et al.  Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase , 1997, Cell.

[17]  S. Dalton,et al.  Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A. Carr,et al.  Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function , 1997, Molecular and General Genetics MGG.

[19]  B. Stillman,et al.  Initiation of chromosomal DNA replication in eukaryotes. Lessons from lambda. , 1994, The Journal of biological chemistry.

[20]  J. Broach,et al.  Genome dynamics, protein synthesis, and energetics , 1991 .

[21]  G. Lucchini,et al.  The DNA polymerase α-primase complex couples DNA replication, cell-cycle progression and DNA-damage response , 1997 .

[22]  C. F. Hardy.  Identification of Cdc45p, an essential factor required for DNA replication. , 1997, Gene.

[23]  L. Johnston,et al.  Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae. , 1991, Nucleic acids research.

[24]  W. L. Fangman,et al.  The localization of replication origins on ARS plasmids in S. cerevisiae , 1987, Cell.

[25]  M. Yanagida,et al.  Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control. , 1994, The EMBO journal.

[26]  J. Diffley,et al.  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication , 1998, Nature.

[27]  Bruce Stillman,et al.  ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex , 1992, Nature.

[28]  C. Wittenberg,et al.  DNA Polymerase ε Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability , 1999 .

[29]  D. Levy,et al.  Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase ε in DNA Replication and the S/M Checkpoint Pathway* , 1998, The Journal of Biological Chemistry.

[30]  S. Elledge,et al.  DNA polymerase ϵ links the DNA replication machinery to the S phase checkpoint , 1995, Cell.

[31]  R. Weinberg,et al.  Identification of a protein associated with p21ras by chemical crosslinking. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[32]  D. Botstein,et al.  Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. , 1982, Genetics.

[33]  R. Roth,et al.  In vivo cross-linking of protein disulfide isomerase to immunoglobulins. , 1987, Biochemistry.

[34]  K. Shirahige,et al.  Regulation of DNA-replication origins during cell-cycle progression , 1998, Nature.

[35]  J. Blow,et al.  The role of MCM/P1 proteins in the licensing of DNA replication. , 1996, Trends in biochemical sciences.

[36]  O. Aparicio,et al.  Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Elledge,et al.  Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. , 1998, Genes & development.

[38]  L. Johnston,et al.  DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Yanagida,et al.  Fission yeast cut5 +, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4 + , 1993, Cell.

[40]  J. Mornon,et al.  From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair , 1997, FEBS letters.

[41]  S. Dalton,et al.  Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit , 1997, Molecular and cellular biology.

[42]  A. Sugino,et al.  Yeast DNA polymerases and their role at the replication fork. , 1995, Trends in biochemical sciences.

[43]  H. Takisawa,et al.  Xenopus Cdc45‐dependent loading of DNA polymerase α onto chromatin under the control of S‐phase cdk , 1998, The EMBO journal.

[44]  M. Sternberg,et al.  Structure of an XRCC1 BRCT domain: a new protein–protein interaction module , 1998, The EMBO journal.

[45]  R. Hamatake,et al.  A third essential DNA polymerase in S. cerevisiae , 1990, Cell.

[46]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[47]  J. Diffley,et al.  Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle , 1995, Journal of Cell Science.

[48]  D. Botstein,et al.  A group of interacting yeast DNA replication genes. , 1991, Genes & Development.

[49]  Peer Bork,et al.  A superfamily of conserved domains in DNA damage‐ responsive cell cycle checkpoint proteins , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[50]  T. Kelly,et al.  SV40 DNA replication. , 1988, The Journal of biological chemistry.

[51]  S. Leem,et al.  Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[52]  B. Stillman,et al.  CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication , 1997, Molecular and cellular biology.