Comparison of the rate of excision of major UV photoproducts in the strands of the human HPRT gene of normal and xeroderma pigmentosum variant cells.
Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts from such patients are extremely sensitive to mutations induced by UV radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (HPRT) gene differs significantly from that seen in normal cells. To determine if this UV hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (CPD) or 6-4 pyrimidine-pyrimidones (6-4s) in that gene, we synchronized XP variant and normal fibroblasts, irradiated them in early G1-phase, 12 or more hours prior to the scheduled onset of S phase, harvested them immediately or after allowing various times for repair, and analyzed the DNA for photoproducts in the HPRT gene, using quantitative Southern blotting. To incise the DNA at CPD, we used T4 endonuclease V; to incise at 6-4s, we first used photolyase and UV365nm to reverse CPD and then UvrABC excinuclease. Excision of CPD was rapid, preferential, and strand-specific, but there was no significant difference in rate between the two kinds of cells. The half life was 4 h in the transcribed strand of the gene and 6.5 h in the nontranscribed strand. For excision of CPD in the genome overall, this value is 12 h. Excision of 6-4s from either strand of the HPRT gene was extremely rapid and preferential in both kinds of cells, with a half life of approximately 30 min. The results indicate that the UV hypermutability of the XP variant cells cannot be caused by slower rates of repair of CPD and/or 6-4s in the target gene for mutagenesis.
Tung
B S
BS
Carcinogenesis Laboratory, Fee Hall, Department of Microbiology, Michigan State University, East Lansing 48824-1316, USA.
McGregor
W G
WG
Wang
Y C
YC
Maher
V M
VM
McCormick
J J
JJ
D015139:Blotting, Southern; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000528:radiation effects; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D011740:Pyrimidine Dimers / Q000378:metabolism*; D011836:Radiation Tolerance / Q000235:genetics*; D014466:Ultraviolet Rays; D014764:Viral Proteins; D014983:Xeroderma Pigmentosum / Q000201:enzymology / Q000235:genetics*
D029968:Escherichia coli Proteins; D011740:Pyrimidine Dimers; D014764:Viral Proteins; C058406:pyrimidine-pyrimidone dimer; D007041:Hypoxanthine Phosphoribosyltransferase; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; C036385:endodeoxyribonuclease uvrABC; D043224:Deoxyribonuclease (Pyrimidine Dimer)