This research aims to understand the factors that regulate DNA repair in mammalian cells. It has been found that DNA damage, especially UV-induced cyclobutane dimers (CPD), are preferentially in the transcribed strand of active gene domains. Current research and concepts are largely based on the premise that transcription and DNA repair may be coupled. Dr. Tang however, have made several findings which lead to different conclusions: 1) there is no strand specificity for the repair of benzo(a)pyrene diol epoxide (BPDE)- and CC-1065-DNA adducts and CPD in the APRT gene even though only one strand of the gene is transcribed, 2)the repair of CPD in a transcriptionally inactive, promoterless APRT gene is the same as in an intact functionally active APRT gene, and 3) the repair of BPDE-DNA adducts in an unamplified DHFR locus is significantly more efficient than in highly amplified loci, and the repair lacks transcribed-strand specificity. To account for these findings they hypothesize that DNA repair is primarily facilitated by chromatin structure of active gene domains and the changes in structure that precede transcription. Dr. Tang reasons that the timing of replication of a gene may determine the binding of the scarce trans-acting transcription factors which could influence the formation of distinctive chromatin structures in these active gene domains. To test this hypothesis they propose to determine the chromatin structures reflected by DNase 1 and restriction enzyme digestion sensitivity and nuclear matrix association of the endogenous, intact as well as the promoterless APRT gene domains. They will also determine the kinetics and strand specificity of CPD repair in APRT gene domains in transfectant cell lines containing single-copy integrations of APRT sequences in different genomic locations; if there are differences in different lines we will then determine the replication time frame of the transfected APRT locus in these cells. They will also test their hypotheses that BPDE binding to nuclear proteins alters chromatin structure and consequently affects the repair of BPDE-DNA adducts; and that the chromatin structure of unamplified and highly amplified DHFR may be different. These experiments will help define the relationships between chromatin structure, timing of gene replication, and the location of a gene in chromosome and their influence on the efficiency of DNA repair.
| Denissenko, M F; Chen, J X; Tang, M S et al. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc Natl Acad Sci U S A 94:3893-8 |
