Differences in the processing of DNA damage in selected genomic domains may account for some of the profound differences seen in the carcinogenic response in different tissues or when different organisms are compared. Our long-term objective is to understand the """"""""fine structure"""""""" of DNA replication and repair by using defined nucleotide sequences containing well-characterized DNA lesions. The T4 bacteriophage endonuclease V is used in a recently developed sensitive assay for ultraviolet (UV) induced pyrimidine dimers in restriction fragments of genes and other domains in the genomes of cultured rodent and human cells. Efforts focus upon studies to learn the rules governing accessibility of pyrimidine dimers, bulky adducts and interstrand crosslinks to repair. Repair will be assessed in the region surrounding the proficiently-repaired dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells to determine boundaries of one of the repairable domains in these generally repair deficient cells. Comparative analyses will be carried out in UV-sensitive CHO mutant cells expressing the cloned denV gene (ie.e., T4 endo V). Methylation levels in genomic regions will be assessed as possible signals for regulation of intragenomic heterogeneity in damage processing. The E. coli UvrABC complex will be used to analyze genomic distribution of bulky adducts produced by N-acetoxyacetylaminofluorene and the psoralens. A sensitive assay based upon renaturability of DNA will be used to monitor introduction and repair of psoralen interstrand cross-links in defined genomic fragments. Repair will be compared in genes that differ in their expression level and function in the cell type under examination. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair accounts for rapid genomic evoluation in rodents. Replication of defined nucleotide sequences in the region around the DHFR gene will be studied to determine whether differential replication occurs in particular damaged genomic domains and whether daughter-strand discontinuities occur in those sequences. The studies should help to interpret the effects of DNA damage upon biological end points such as survival, mutagenesis, and transformation.
Showing the most recent 10 out of 19 publications
