The goal of the proposal is to understand the mechanism by which an origin of DNA replication is converted into a replication fork and how the proteins at the fork are organized to coordinate the synthesis of the leading and lagging strands. The underlying assumption, which, while plausible, remain to be proved, is that there is a super assembly of proteins at the replication fork and that it functions like a machine whose moving parts are proteins. Wha is less certain is whether there is a single complex assembled at origins that changes conformation in a concerted manner to effect each stage of fork progression or if there are a number of smaller machines each assigned a different specific task and only transiently associated with the fork. To attack such a complex problem requires dividing it into partial reactions. In the coming granting period the investigator will study in depth the Dna2 helicase and DNA polymerase epsilon, with a focus on the dynamics of protein/protein interactions of these key components. They would use yeast as model system to allow a combined genetic and biochemical approach. In the past granting period, the investigators described a helicase essential for chromosomal DNA replication. It was discovered that it interacted with FEN-1, a protein involved in Okazaki fragment processing in SV40 in vitro DNA replication. Further studies of Dna2 have the goal of determining whether the Dna2 helicase plays a role in maturation of the lagging strand in yeast cells and if so, does it have a catalytic role or an architectural/structural role. It should go without saying that to understand the assembly, activation, and movement of the replication fork, one must understand the DNA polymerase. This grant has supported studies of the yeast DNA polymerases for many years. Recently the investigators have focused on pol epsilon. They have used site-directed mutagenesis in an analysis of the C-terminal half of the catalytic core polypeptide, which may have a dual role in DNA replication and sensing DNA damage. The investigators will continue to determine the protein/protein contacts in the pol epsilon holoenzyme and to determine the role of individual subunits. They will use chromatin crosslinking to study the dynamics of interactions between the three DNA polymerases at replication origins and forks, ultimately to get at the question of which polymerase copie which strand, taking advantage of all the polymerase mutants and clones produced under this grant.

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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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California Institute of Technology
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Jaszczur, Malgorzata; Rudzka, Justyna; Kraszewska, Joanna et al. (2009) Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae. Mutat Res 669:27-35
Lou, Huiqiang; Komata, Makiko; Katou, Yuki et al. (2008) Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Mol Cell 32:106-17
Jaszczur, Malgorzata; Flis, Krzysztof; Rudzka, Justyna et al. (2008) Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae. Genetics 178:633-47
Reis, Clara C; Campbell, Judith L (2007) Contribution of Trf4/5 and the nuclear exosome to genome stability through regulation of histone mRNA levels in Saccharomyces cerevisiae. Genetics 175:993-1010
Boronat, Susanna; Campbell, Judith L (2007) Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55. Mol Cell Biol 27:1158-71
Masuda-Sasa, Taro; Polaczek, Piotr; Campbell, Judith L (2006) Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation. J Biol Chem 281:38555-64
Masuda-Sasa, Taro; Imamura, Osamu; Campbell, Judith L (2006) Biochemical analysis of human Dna2. Nucleic Acids Res 34:1865-75
Stewart, Jason A; Campbell, Judith L; Bambara, Robert A (2006) Flap endonuclease disengages Dna2 helicase/nuclease from Okazaki fragment flaps. J Biol Chem 281:38565-72
Budd, Martin E; Reis, Clara C; Smith, Stephanie et al. (2006) Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol Cell Biol 26:2490-500
Budd, Martin E; Tong, Amy Hin Yan; Polaczek, Piotr et al. (2005) A network of multi-tasking proteins at the DNA replication fork preserves genome stability. PLoS Genet 1:e61

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