The overall goal of this grant is to study CDC6 gene function and its cell cycle regulation. Early genetic studies revealed that yeast temperature-sensitive mutant, cdc6, is defective in the late G1 and S phase boundary, and that its function may be, either directly or indirectly, involved in DNA replication. We have previously isolated and characterized the CDC6 gene. We have also demonstrated that CDC6 mRNA and nuclear entry of CDC6 protein are cell cycle dependent. In this submission period, we have found that the GTS::CDC6 fusion protein has a DNA-dependent ATPase activity. In this proposal, we will use a combined molecular genetic and biochemical approach to investigate the biological function(s) of CDC6 and its cell cycle regulation. Initially, we will identify the cellular components which interact with CDC6 gene products. In order to accomplish this, we will isolate and characterize the suppressor(s) to establish a linked pathway between CDC6 and other related genes in the cell cycle progression. We will also use a molecular genetic approach, the 2-hybrid system, to identify the protein component(s) which interact with CDC6 in vivo. The results will complement our genetic studies, providing additional information to establish a pathway of cell cycle progression. Secondly, we will explore the gene function(s) of CDC6 via the biochemical approach. We will characterize the purified CDC6 protein by investigating its nucleotide-binding, ATPase, and/or GTPase activities. Based on the sequence motif, we will also detect possible helicase activity. Point-mutated cdc6 proteins will also be purified to complement the above biochemical studies. It is anticipated that the results will shed light on the biological function(s) of CDC6. We expect that the investigation of the yeast CDC6 gene and its suppressors will lead to an understanding of the functions of corresponding genes in other eukaryotes, and may provide us with useful information to disclose the profound control mechanism of the cell division cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048492-02
Application #
2185959
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1993-08-01
Project End
1997-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
094878337
City
Los Angeles
State
CA
Country
United States
Zip Code
90027
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Feng, L; Hu, Y; Wang, B et al. (2000) Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif. DNA Cell Biol 19:447-57
Wang, B; Feng, L; Hu, Y et al. (1999) The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association. J Biol Chem 274:8291-8
Jong, A Y; Yu, K; Zhou, B et al. (1998) A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5:62-8
Feng, L; Wang, B; Jong, A (1998) Saccharomyces cerevisiae Cdc6 stimulates Abf1 DNA binding activity. J Biol Chem 273:1298-302
Huang, S H; Jong, A (1997) Expression and preparation of fusion proteins from recombinant lambda gt11 phages. Methods Mol Biol 69:241-5
Elsasser, S; Lou, F; Wang, B et al. (1996) Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases. Mol Biol Cell 7:1723-35
Jong, A Y; Wang, B; Zhang, S Q (1995) Pulsed field gel electrophoresis labeling method to study the pattern of Saccharomyces cerevisiae chromosomal DNA synthesis during the G1/S phase of the cell cycle. Anal Biochem 227:32-9