XPA protein alters the specificity of ultraviolet light-induced mutagenesis in vitro.

Studies of ultraviolet (UV) light mutagenesis have demonstrated mutations at common sites in the target genes of shuttle vector plasmids replicated in cultured cells or by cellular extracts. The reasons for the specific pattern of mutagenesis are largely unknown. We have examined the specificity of UV-induced mutagenesis by replicating plasmid pLS189, irradiated with 40 J/m(2) UVC or unirradiated, in either xeroderma pigmentosum group A (XP-A) or HeLa cellular extracts. The XP-A extract displayed slightly lower replication ability, but produced a higher mutant frequency, compared to that of HeLa extract. Use of irradiated plasmid inhibited replication by an average of 63% and increased the mutant frequency by an average of 16.7-fold. Analysis of mutation spectra revealed nonrandom patterns of mutagenesis that differed significantly between HeLa and XP-A extracts. In comparison to HeLa extract, replication in XP-A extract resulted in lower frequencies of GC --> AT transitions and tandem double-base substitutions, and a higher frequency of deletions. Replication in HeLa extract produced hotspots at positions 100, 108, and 156 that were not produced by XP-A extract. Furthermore, XP-A extract produced hotspots at positions 124, 133, and 164, sites not characteristic of previous UV-induced mutagenesis studies using XPA-expressing cells. Addition of purified XPA protein to reactions containing XP-A extract altered each of these parameters, including loss of the hotspots at positions 124 and 133, to yield a more HeLa-like spectrum. These results indicate a previously uncharacterized role of the XPA protein in influencing the specificity of UV-induced mutagenesis during DNA replication.