The goal of this research is to identify the mechanisms by which the S-phase DNA damage checkpoint responses arrest DNA replication. These checkpoints are the way cells temporarily stop passage through the cell cycle to allow time for repair of DNA damage, prior to cell division. If these checkpoints don't act appropriately, there are various possible outcomes. Minor DNA damage can lead to permanent mutations in the genome. Greater DNA damage can lead to chromosomal breakage, rearrangement, translocations, and even catastrophic cell division. The principal investigator's preliminary studies have shown that he can use an SV4O in vitro DNA replication system, which is supported predominantly by human cell extracts, to biochemically investigate S phase DNA damage dependent checkpoints. Pretreatment of cultured human cells with DNA damaging agents leads to inhibition of in vitro SV4O DNA replication that parallels the inhibition of chromosomal DNA replication in vivo. As model drugs, the principal investigator has chosen to use two anti-cancer chemotherapeutics. He has shown that these two drugs trigger different mechanisms for arresting DNA replication, adozelesin inactivates a known cellular DNA replication protein, RPA, while bizelesin induces the presence of a trans-acting DNA replication inhibitor. The goals of this proposal are to understand how adozelesin results in the inactivation of RPA, and to identify the trans-inhibitor induced by bizelesin, identify the replication protein that is the target of this trans-inhibitor, and to determine how this second mechanism results in the inhibition of DNA replication. During their progression, most forms of cancer have lost one or more of their DNA-damage checkpoint responses. This likely explains why most cancer therapies generally destroy cancer cells through catastrophic cell division. Elucidating these DNA-damage dependent checkpoint pathways and understanding the mechanisms of how they work, will ultimately aid in the design of better anti-cancer therapeutics, and will provide information that will allow for improved therapeutic strategies for particular tumors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089259-02
Application #
6489408
Study Section
Biochemistry Study Section (BIO)
Program Officer
Spalholz, Barbara A
Project Start
2001-01-12
Project End
2005-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
2
Fiscal Year
2002
Total Cost
$262,795
Indirect Cost
Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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Masih, Prerna Jasmine; Kunnev, Dimiter; Melendy, Thomas (2008) Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA). Nucleic Acids Res 36:67-75
Liu, Jen-Sing; Kuo, Shu-Ru; Melendy, Thomas (2006) DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation. J Cell Biochem 99:1452-62
Liu, Jen-Sing; Kuo, Shu-Ru; Melendy, Thomas (2006) Phosphorylation of replication protein A by S-phase checkpoint kinases. DNA Repair (Amst) 5:369-80
Tu, Lan Chun; Melendy, Thomas; Beerman, Terry A (2004) DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic. Mol Cancer Ther 3:577-85
Cao, Pei-rang; McHugh, Mary M; Melendy, Thomas et al. (2003) The DNA minor groove-alkylating cyclopropylpyrroloindole drugs adozelesin and bizelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells. Mol Cancer Ther 2:651-9
Liu, Jen-Sing; Kuo, Shu-Ru; Melendy, Thomas (2003) Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents. Mutat Res 532:215-26
Liu, Jen-Sing; Kuo, Shu-Ru; Beerman, Terry A et al. (2003) Induction of DNA damage responses by adozelesin is S phase-specific and dependent on active replication forks. Mol Cancer Ther 2:41-7
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Melendy, T; Li, R (2001) Chromatin remodeling and initiation of DNA replication. Front Biosci 6:D1048-53