The long-term objective of this proposal is to understand the cellular processes that regulate high-fidelity eukaryotic DNA replication. Low fidelity replication and error-prone post-replication DNA repair can result in chromosomal aneuploidy and mutations, which can cause aging, cancer and birth defects. This proposal will employ molecular genetic analysis to identify the regulatory molecules of these processes using the single- celled eukaryote, Saccharomyces cerevisiae as a model system. This combined genetic and molecular approach will focus on a number of important cell cycle protein kinases, which include DDK (Cdc7-Dbf4), CDK (cyclin-dependent kinase) and the Rad53 checkpoint kinase.
Three specific aims are proposed.
In aim #1, we will elucidate the function and regulation of the MCM DNA helicase by investigating both the N- terminal and C- terminal ? (Beta)-fingers and the N-terminal region A domain regulated by DDK. Our hypothesis is that the N- terminal ?-fingers are important for binding origins and the C-terminal fingers are important for helicase translocation or movement along the DNA. Furthermore, we propose that phosphorylation of the MCM complex by DDK produces a structural change in the A domain of Mcm5 resulting in helicase activation. Our system relies on atomic structural and biochemical in vitro studies of the conserved Archaeal MCM helicases and molecular genetic in vivo studies of DNA replication in yeast.
In aim 2, we will investigate a novel role of Rad53 in DNA Replication. Our current hypothesis is that Rad53 regulates proteins important for the initiation of DNA replication and in chromatin structure. This role is independent of Rad53 protein's checkpoint function. In this aim, we will continue to exploit our unique cdc7-mcm5-rad53-histone H3/H4 "interactome" genetic system to identify more interacting proteins and will also analyze changes in chromatin in our mutants.
In aim 3, we investigate the fidelity of DNA replication and the regulation of mutagenesis by studying the role of DDK in TLS (trans-lesion synthesis). Our studies have shown that DDK regulates both spontaneous and induced mutagenesis by DNA polymerase ? (zeta). Our hypothesis is that DDK acts as a "chromatin loader" of Rev7, an important accessory of the Rev3 error-prone DNA polymerase ?. DDK may also be important for replication restart after DNA damage and TLS. A combination of mutational analysis, whole-genome genetic screens, ChIP (chromatin immuno-precipitation), and affinity/antibody isolation will be used to test many of these hypotheses. Our studies have significance for human disease as the human homologues of Rad53 (Chk2) and the TLS polymerase Pol? (eta-XPV) are mutated in the familial cancer-predisposing Li-Fraumeni and Xeroderma pigmentosum variant syndromes, respectively.

Public Health Relevance

Because the proposed studies focus on the fidelity of chromosome replication and the production of genetic mutations, they have relevance to human health with respect to cancer, aging and birth defects. Defects in the several of the human genes studied in this yeast model system are known to increase cancer risk.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035078-22
Application #
8245889
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Reddy, Michael K
Project Start
1985-09-06
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
22
Fiscal Year
2012
Total Cost
$316,002
Indirect Cost
$107,731
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Brandão, Luis N; Ferguson, Rebecca; Santoro, Irma et al. (2014) The role of Dbf4-dependent protein kinase in DNA polymerase ?-dependent mutagenesis in Saccharomyces cerevisiae. Genetics 197:1111-22
Ramey, Christopher J; Sclafani, Robert A (2014) Functional conservation of the pre-sensor one beta-finger hairpin (PS1-hp) structures in mini-chromosome maintenance proteins of Saccharomyces cerevisiae and archaea. G3 (Bethesda) 4:1319-26
Zhong, Yuan; Nellimoottil, Tittu; Peace, Jared M et al. (2013) The level of origin firing inversely affects the rate of replication fork progression. J Cell Biol 201:373-83
Chien, Chia-Yi; Chen, Bo-Ruei; Chou, Chen-Kung et al. (2009) The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett 583:2281-6
Leon, Ronald P; Tecklenburg, Marianne; Sclafani, Robert A (2008) Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae. Genetics 179:1757-68
Sclafani, R A; Holzen, T M (2007) Cell cycle regulation of DNA replication. Annu Rev Genet 41:237-80
Hoang, Margaret L; Leon, Ronald P; Pessoa-Brandao, Luis et al. (2007) Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae. Mol Cell Biol 27:7594-602
Dohrmann, Paul R; Sclafani, Robert A (2006) Novel role for checkpoint Rad53 protein kinase in the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Genetics 174:87-99
Sclafani, Robert A; Fletcher, Ryan J; Chen, Xiaojiang S (2004) Two heads are better than one: regulation of DNA replication by hexameric helicases. Genes Dev 18:2039-45
Pessoa-Brandao, Luis; Sclafani, Robert A (2004) CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae. Genetics 167:1597-610

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