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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM009901-25
Application #
3268067
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1978-07-01
Project End
1991-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
25
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Hanawalt, Philip C (2013) The awakening of DNA repair at Yale. Yale J Biol Med 86:517-23
Hanawalt, P C (1991) Heterogeneity of DNA repair at the gene level. Mutat Res 247:203-11
Ho, L; Hanawalt, P C (1991) Gene-specific DNA repair in terminally differentiating rat myoblasts. Mutat Res 255:123-41
Hanawalt, P C (1990) Selective DNA repair in active genes. Acta Biol Hung 41:77-91
Hanawalt, P C (1990) Selective DNA repair in expressed genes in mammalian cells. Prog Clin Biol Res 340A:213-22
Defais, M; Lesca, C; Monsarrat, B et al. (1989) Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA. J Bacteriol 171:4938-44
Hanawalt, P C (1989) Preferential repair of damage in actively transcribed DNA sequences in vivo. Genome 31:605-11
Ho, L; Bohr, V A; Hanawalt, P C (1989) Demethylation enhances removal of pyrimidine dimers from the overall genome and from specific DNA sequences in Chinese hamster ovary cells. Mol Cell Biol 9:1594-603
Ganesan, A K; Hunt, J; Hanawalt, P C (1988) Temperature dependent survival of UV-irradiated Escherichia coli K12. Mol Gen Genet 214:198-203
Bohr, V A; Hanawalt, P C (1987) Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene. Carcinogenesis 8:1333-6

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