Fine-mapping of DNA damage and repair in specific genomic segments.
The susceptibility of various genomic regions to DNA damage and repair is heterogeneous. While this can be related to factors such as primary sequence, physical conformation, and functional status, the exact mechanisms involved remain unclear. To more precisely define the key features of a genomic region targeted for these processes, a useful tool would be a method for fine-mapping gene-specific DNA damage and repair in vivo. Here, a polymerase chain reaction-based assay is described for measuring DNA damage and repair in small (less than 500 bp) genomic segments of three transcriptionally active but functionally distinct loci (rearranged immunoglobulin heavy chain variable region [Ig VDJ], low-density lipoprotein receptor gene, and N-ras proto-oncogene) in human tonsillar B lymphocytes. Analysis of ultraviolet (254 nm)-induced DNA damage revealed single-hit kinetics and a similar level of sensitivity (D50% approximately 6000 joule/m2) in all three regions, indicating that a single photoproduct was sufficient to fully block PCR amplification. A similar time period per unit length was required for repair of this DNA damage (average t1/2 per fragment length = 23.5 seconds per bp). DNA damage and repair was also detectable with the base adducting agent, 4-nitroquinoline-1-oxide. However, in this case IgVDJ differed from segments within the other two loci by its relative inaccessibility to alkylation. This assay thus permits high-resolution mapping of DNA damage and repair activity.
Govan
H L
HL
Department of Pathology, UCLA School of Medicine 90024-1732.
Valles-Ayoub
Y
Y
Braun
J
J
D001402:B-Lymphocytes; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D005803:Genes, Immunoglobulin; D011905:Genes, ras; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D014066:Palatine Tonsil / Q000166:cytology; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D011519:Proto-Oncogenes; D011973:Receptors, LDL / Q000235:genetics; D012044:Regression Analysis; D014466:Ultraviolet Rays
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011973:Receptors, LDL; D004247:DNA