Cell Cycle-Dependent Strand Bias for UV-Induced Mutations in the Transcribed Strand of Excision Repair-Proficient Human Fibroblasts but Not in Repair-Deficient Cells

Abstract

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G₁ phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G₁ and the repairproficient mutants from S, ~62% were G · C→A T. In the repair-proficient mutants from G₁, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G₁, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; ~75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G₁-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.