While considerable attention has been given to mapping sites of carcinogen-induced mutations in a number of cancer related genes, less is known about the distribution of the original DMA damage that might have given rise to mutagenic lesions. The goal of this R21 project is to develop and validate methods that would detect sites of DNA damage and allow us to determine the distribution of such lesions both globally and at specific locations in genomic DNA. It is our hypothesis that the distribution of DNA damage is not random, but is influenced by the structure and functional properties of genomic DNA. To test this hypothesis, we need to demonstrate that the methods that we are developing will enable us to visualize the distribution of DNA damage sites and relate the damage sites to specific genomic locations. Once we have demonstrated the general feasibility of this approach, we will perform experiments to evaluate the distribution of DNA damage in specific functional targets. We have demonstrated the ability to perform each of the following research steps necessary for the proposed analysis: (1) We have been able to prepare DNA for fiber analysis using molecular combing; (2) We localized a specific DNA sequence among an excess of genomic DNA using fluorescence in situ hybridization (FISH) on combed DNA fibers; (3) We visualized the distribution of apurinic/apyrimidinic (AP) sites in DNA using either an aldehyde reactive probe tagged with biotin or a fluorescent probe; (4) We were able to visually identify AP sites within regions undergoing replication; and (5) We identified specific regions undergoing replication (all described in the Preliminary Studies section). In this study, we propose to show that it is feasible to combine these techniques in a novel way to establish whether certain areas of the genome, for example sites that are being replicated, are more sensitive to DNA damage than other genomic regions (or, the same sites in the absence of DNA replication). To accomplish this goal, we will determine the sensitivity of detection of AP sites in combed DNA using DNA fibers with a known number of AP sites. We will then determine the background distribution of AP sites in untreated and treated DNA. Finally, we will determine whether it is possible to detect visual signals from all three techniques (AP sites, FISH, DNA counterstain) in the same experimental samples. ? ? ASSESSMENT: ? ? ?
| Kaufman, David G; Cohen, Stephanie M; Chastain, Paul D (2011) Temporal and functional analysis of DNA replicated in early S phase. Adv Enzyme Regul 51:257-71 |
| Luke, April M; Chastain, Paul D; Pachkowski, Brian F et al. (2010) Accumulation of true single strand breaks and AP sites in base excision repair deficient cells. Mutat Res 694:65-71 |
| Chastain 2nd, Paul D; Nakamura, Jun; Rao, Shangbang et al. (2010) Abasic sites preferentially form at regions undergoing DNA replication. FASEB J 24:3674-80 |
