Many therapeutic alkylating agents including the nitrogen mustards, chloroethylnitrosoureas, and methyl methane sulfonates form interstrand cross-links with their DNA targets. These cross-links if left unrepaired will prevent DNA strand separation and normal mitosis, interfere with transcription, and induce apoptosis. Increasing evidence indicates that such cross-links are recognized by cells, and this recognition can mediate either repair of the damage and survival, or accelerated death of the cell. The long term goal of this revised application is to correlate the structure of DNA that contains interstrand cross-links with the ability of cells to repair these cross-links. To achieve this goal methods will be developed to synthesize on controlled pore glass supports, short DNA duplexes that contain interstrand cross-links or cross-link mimics. These cross-linked duplexes will be incorporated into plasmid DNA. The cross-linked DNAs will be tested for their ability to serve as substrates for repair enzymes found in extracts from human tumor cell lines that are either susceptible or resistant to the action of therapeutic alkylating agents. Electorphoretic gel mobility shift assays and cross-linked DNA affinity columns will be used to characterize and isolate proteins and enzymes involved in the repair process. The proposed studies are expected to lead to a better understanding of the mechanisms involved in the repair of interstrand cross-links and could lead to the development of inhibitors of interstrand cross-link repair that could be used to enhance the efficacy of therapeutic alkylating agents in the treatment of tumors.
| 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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