The long term objective of our research project is to gain insight into the regulatory mechanism for DNA replication initiation in eukaryotes using yeast as our model system. The regulation of DNA replication initiation is an important aspect of the regulation of cell growth. The goal of this proposal is fundamental in nature but may have useful applications to the control of the growth of tumorigenic cells. In yeast, specific DNA sequences known as autonomously replicating sequences (ARSs) have been isolated. These ARSs are believed to be the initiation sites for DNA replication on chromosomes. We examined the mechanism of replication initiation at these sites using three different approaches. First, we identified genes that affect the function of ARSs on minichromosomes by the isolation of minichromosome maintenance defective (Mcm) mutants. We then cloned and sequenced these genes. We also examined the biochemical function and properties of the products of these MCM genes. Second, we isolated proteins that interact directly and specifically with ARSs. Third, we analyzed the functional sequence of ARSs with reference to these mutants and the ARS-binding proteins (ABPs).
Our specific aims for the next funding period will be as follows: (1) We will continue to study the biochemical and biological functions of three MCM gene products, MCM1 gene products, MCM1, MCM2 and MCM3. Special attention will be given to the analysis of the MCM1 gene product which has been shown to be a transcriptional regulator. The relationship between its role in minichromosome maintenance and transcriptional activation of mating-type specific genes will be investigated. (2) We will further define the sites of action of the MCM gene products at ARSs by direct DNA binding studies. Deletion analysis of ARSs in the mcm mutants has already implicated that the site of action of the MCM gene products is on the 5' side of the ARS consensus sequence. (3) We will look for additional ABPs that would bind to the 5' side of the ARS consensus sequence.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034190-12
Application #
3284756
Study Section
Molecular Biology Study Section (MBY)
Project Start
1978-04-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Liachko, Ivan; Tye, Bik K (2005) Mcm10 is required for the maintenance of transcriptional silencing in Saccharomyces cerevisiae. Genetics 171:503-15
Douglas, Nancy L; Dozier, Samantha K; Donato, Justin J (2005) Dual roles for Mcm10 in DNA replication initiation and silencing at the mating-type loci. Mol Biol Rep 32:197-204
Chang, Victoria K; Donato, Justin J; Chan, Clarence S et al. (2004) Mcm1 promotes replication initiation by binding specific elements at replication origins. Mol Cell Biol 24:6514-24
Tye, Bik K; Chang, Victoria K (2004) Dual functional regulators coordinate DNA replication and gene expression in proliferating cells. Front Biosci 9:2548-55
Sawyer, Sara L; Cheng, Irene H; Chai, Weihang et al. (2004) Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae. J Mol Biol 340:195-202
Fitch, Michael J; Donato, Justin J; Tye, Bik K (2003) Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1. J Biol Chem 278:25408-16
Christensen, Tim W; Tye, Bik K (2003) Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation. Mol Biol Cell 14:2206-15
Chang, Victoria K; Fitch, Michael J; Donato, Justin J et al. (2003) Mcm1 binds replication origins. J Biol Chem 278:6093-100
Lei, Ming; Cheng, Irene H; Roberts, Louis A et al. (2002) Two mcm3 mutations affect different steps in the initiation of DNA replication. J Biol Chem 277:30824-31
Yu, Xiong; VanLoock, Margaret S; Poplawski, Andrzej et al. (2002) The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings. EMBO Rep 3:792-7

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