The goal of our research is to identify and characterize the machinery involved in eukaryotic DNA replication. We focus on the isolation of the individual proteins and protein complexes required for DNA synthesis and define their role in this critical macromolecular process. All growing cells must duplicate their DNA accurately and perform this feat once and only once per cell cycle. Events that interfere with these requirements can lead to cell death or altered proliferation that result in cancers. At present, many steps involved in supporting replication and its control are either unknown or poorly characterized. We plan to extend our previous work on various key replication proteins and their complexes that support the activation of initiation complexes required for the elongation of DNA chains. For this purpose, we will study: a) the role of a complex, containing Cdc45, the MCM2-7 complex and GINS (CMG) which contains DNA helicase activity and may contribute important to the unwinding reaction at the replication fork;b) the interaction between GINS and the Pol 1-primase complex which markedly stimulates its DNA polymerase activity;c) the properties of cloned Pol5 and its interaction with PCNA required for its processive elongation of primed DNA templates. We have also observed functional interactions of GINS with Pol5 as well as Pol4. We plan to examine these effects further and evaluate whether the CMG complex does the same. Coupled replication and unwinding reactions will be carried out which may contribute to strand selection;d) We plan to explore the role of the Cdc7 kinase on the activity and formation of the CMG complex. In vivo, this kinase was shown to influence the interaction of Cdc45 with the MCM2-7 complex. We will further characterize a novel interaction recently detected between the Cdc7 kinase complex and the cohesin loader complex Scc2/Scc4. The requirements for this interaction and its consequences will be investigated. All cells must make identical copies of their DNA to survive. Our studies are directed at how these copies are made and regulated. We plan to isolate the proteins involved in copying DNA and determine how they function. Results from these studies will have a direct impact on genetic diseases and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034559-27
Application #
7740854
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Hagan, Ann A
Project Start
1984-07-01
Project End
2011-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
27
Fiscal Year
2010
Total Cost
$777,648
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Cho, Won-Ho; Kang, Young-Hoon; An, Yun-Young et al. (2013) Human Tim-Tipin complex affects the biochemical properties of the replicative DNA helicase and DNA polymerases. Proc Natl Acad Sci U S A 110:2523-7
Kang, Young-Hoon; Farina, Andrea; Bermudez, Vladimir P et al. (2013) Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase. Proc Natl Acad Sci U S A 110:19760-5
Kang, Young-Hoon; Galal, Wiebke Chemnitz; Farina, Andrea et al. (2012) Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis. Proc Natl Acad Sci U S A 109:6042-7
Chemnitz Galal, Wiebke; Pan, Miao; Giulian, Gary et al. (2012) Formation of dAMP-glycerol and dAMP-Tris derivatives by Thermococcus kodakaraensis DNA primase. J Biol Chem 287:16220-9
Yardimci, Hasan; Wang, Xindan; Loveland, Anna B et al. (2012) Bypass of a protein barrier by a replicative DNA helicase. Nature 492:205-9
Fu, Yu V; Yardimci, Hasan; Long, David T et al. (2011) Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146:931-41
Li, Zhuo; Pan, Miao; Santangelo, Thomas J et al. (2011) A novel DNA nuclease is stimulated by association with the GINS complex. Nucleic Acids Res 39:6114-23
Ladner, Jane E; Pan, Miao; Hurwitz, Jerard et al. (2011) Crystal structures of two active proliferating cell nuclear antigens (PCNAs) encoded by Thermococcus kodakaraensis. Proc Natl Acad Sci U S A 108:2711-6
Im, Jun-Sub; Ki, Sang-Hee; Farina, Andrea et al. (2009) Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins. Proc Natl Acad Sci U S A 106:15628-32
Vijayakumar, Sangeetha; Dziegielewska, Barbara; Levin, David S et al. (2009) Phosphorylation of human DNA ligase I regulates its interaction with replication factor C and its participation in DNA replication and DNA repair. Mol Cell Biol 29:2042-52

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