Many clinically important cancer chemotherapeutic agents are bifunctional alkylating reagents that produce lethal interstrand cross-links in tumor cell DNA Tumors can develop resistance to such agents by repairing these cross-links. The goal of this project is to understand how such interstrand cross-links are recognized and subsequently repaired by cells, particularly by human tumor cells that are either susceptible or resistant to therapeutic alkylating agents. The project has four specific aims. In the first specific aim, short DNA duplexes that contain therapeutically relevant interstrand cross-links or cross-link mimics will be prepared on an automated DNA synthesizer. These will include N4C-alkyI-N4C, N3C-alkyI-N3C, N1G-ethyI-N3C, N7G-alkyl -N7G and O6G-alkyI-O6G cross-links. The second specific aim will study the effects of these cross-links on the structures of the DNA duplexes in which they reside. Thermal denaturation, CD spectroscopy and gel mobility shift experiments will be used to characterize the global structures of the cross-linked duplexes, while high resolution NMR and molecular dynamics will be used to characterize the atomic structures and dynamics of the duplexes. The third specific aim will examine the interactions of proteins that are involved in DNA repair with larger DNA duplexes and plasmid DNA that contain a single interstrand cross-link.
This specific aim will also assess the ability of nuclear extracts derived from mammalian cells and mutants deficient in selected repair proteins to repair the cross-links in vitro. The fourth specific aim will examine the repair of plasmid DNA or yeast chromosomal DNA, which contains a single defined interstrand cross-link, in E. coli, Saccharomyces cerevisiae and mammalian cells and human tumor cell lines. These studies will focus on characterizing the structural and dynamic perturbations induced in the DNA duplexes by a defined set of cross-links of differing geometries. Combining this information with our knowledge of how the crosslinks are repaired will allow us to identify what changes in duplex structure or dynamics are responsible for the recognition of these cross-link lesions in DNA Such information will contribute to our basic understanding of tumor cell resistance to therapeutic alkylating agents and could lead to the development of inhibitors of interstrand cross-link repair, which could be used to enhance the efficacy of therapeutic alkylating agent in the treatment of cancer.
| Miller, Paul S (2011) Syntheses of DNA duplexes that contain a N?C-alkyl-N?C interstrand cross-link. Curr Protoc Nucleic Acid Chem Chapter 5:Unit5.10 |
| Friedman, Joshua I; Jiang, Yu Lin; Miller, Paul S et al. (2011) Unique dynamic properties of DNA duplexes containing interstrand cross-links. Biochemistry 50:882-90 |
| Hlavin, Erica M; Smeaton, Michael B; Noronha, Anne M et al. (2010) Cross-link structure affects replication-independent DNA interstrand cross-link repair in mammalian cells. Biochemistry 49:3977-88 |
| Hlavin, Erica M; Smeaton, Michael B; Miller, Paul S (2010) Initiation of DNA interstrand cross-link repair in mammalian cells. Environ Mol Mutagen 51:604-24 |
| Smeaton, Michael B; Hlavin, Erica M; Noronha, Anne M et al. (2009) Effect of cross-link structure on DNA interstrand cross-link repair synthesis. Chem Res Toxicol 22:1285-97 |
| Smeaton, Michael B; Hlavin, Erica M; McGregor Mason, Tracey et al. (2008) Distortion-dependent unhooking of interstrand cross-links in mammalian cell extracts. Biochemistry 47:9920-30 |
| Mason, Tracey McGregor; Smeaton, Michael B; Cheung, Joyce C Y et al. (2008) End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion. Bioconjug Chem 19:1064-70 |
| Smeaton, Michael B; Miller, Paul S; Ketner, Gary et al. (2007) Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining. Nucleic Acids Res 35:e152 |
| Swenson, Matthew C; Paranawithana, Shanthi R; Miller, Paul S et al. (2007) Structure of a DNA repair substrate containing an alkyl interstrand cross-link at 1.65 A resolution. Biochemistry 46:4545-53 |
| Noll, David M; Mason, Tracey McGregor; Miller, Paul S (2006) Formation and repair of interstrand cross-links in DNA. Chem Rev 106:277-301 |
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