Mutation spectrum in UVB-exposed skin epidermis of a mildly-affectedXpg-deficient mouse

Abstract

A C-terminal 183 amino acid-truncated mutation of the mouse Xpg gene (XpgDeltaex15) gives rise to a partial deficiency in nucleotide excision repair in homozygously affected cells. We studied the effect of this mutation on UVB-induced mutagenesis in mouse skin, using transgenic mice harboring lambda-phage-based bacterial lacZ genes as a mutational reporter. UVB increased the lacZ mutant frequency in the epidermis moderately in the homozygous mutant mice, but significantly higher than in the wild-type or the heterozygous mice, whereas background mutant frequencies were not appreciably different among the three mouse genotypes. Ninety-eight lacZ mutant sequences isolated from the UVB-exposed epidermis of the XpgDeltaex15-homozygous mice were analyzed and compared with mutant sequences from the wild-type mice. The spectra of the mutations in the two mouse genotypes were not significantly different, and they were highly UV-specific. There were frequent C --> T transitions at dipyrimidine sites and several CC --> TT tandem mutations, although the UV-specific mutations occurred more frequently at CpG sites in the mutant mice. The distribution of the mutations observed in the lacZ transgene and the preferred sequence context of the UV-specific C --> T mutations (5'-TC-3' > 5'-CC-3' > 5'-CT-3') in the Xpg-mutant mice were similar to those found in the wild-type mice. Despite these similarities, we detected a previously unrecognized type of the UV-induced mutation only in the Xpg mutant (6/98 in the mutation spectrum of the mutant vs. 0/76 in the wild-type; P = 0.035), which is characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We propose that this putative new class of mutation, which we refer to as a "triplet mutation", is characteristic of UV-induced mutation in an excision-repair-deficient background.

Mutation spectrum in UVB‐exposed skin epidermis of Xpa‐knockout mice: Frequent recovery of triplet mutations

“…Although the existence of UV-induced triplet mutations had not been recognized until our study using Xpg mutant mice [Wang et al, 2006], several examples of these mutations can be found among studies of in vivo UV-induced mutation reported in the literature. The greatest number was reported in a study using another transgenic mouse with the same Xpa-null mutation as ours [Murai et al, 2000], where 13 mutations that can be classified as triplet mutations (although the authors listed them as a part of double mutations) were detected in a transgenic bacterial reporter gene from UVB-exposed skin epidermis (13/256).…”

“…TT, three-base tandem/alternate substitutions, and a complex mutation; see Tables I, III, and V). A low frequency of triplet mutations was detected in our previous study of the UVB-induced mutation spectrum in a Xpg mutant mouse, which had a C-terminal 183-amino acid-deletion of the Xpg gene product and showed a partial deficiency in NER [Wang et al, 2006]. In this study, we defined the triplet mutation as a complex mutation characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine.…”

“…TT twobase tandem and the three-base tandem/alternate mutations had in common a complex alteration of two or three base substitutions within a three nucleotide context that included a dipyrimidine (in the case of two-base tandem substitutions, a neighboring nucleotide on either side of the affected dinucleotide was included in the three nucleotide context; see the 18th to 5th mutants except the 16th from the bottom in Table I). This type of mutation has been identified recently as a unique type of UV-induced mutation, and is referred to as a 'triplet mutation' [Wang et al, 2006]. According to the definition of triplet mutation, in which base substitutions can be replaced with single-base frameshifts, the complex mutation (the 16th mutant from the bottom in Table I) also qualifies as a triplet mutation.…”

“…Furthermore, we found a large number of triplet mutations, defined as a mutation associated with multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. A small number of these mutations was recently detected in a study of the mutation spectrum induced in UVB-exposed epidermis from a mildly-affected Xpg mutant mouse [Wang et al, 2006]. We discuss the significance of triplet mutation in terms of the effect of NER deficiency on UV-induced mutagenesis.…”

“…DNA sequence analysis of the mutant lacZ genes was performed as described [Wang et al, 2006]. Briefly, each mutant lacZ gene was amplified by PCR as four overlapping 1-kb fragments (lac1 to lac4) that cover the entire coding region, using four sets of primers (NUlac1 and NLlac1, NUlac2 and NLlac2, NUlac3 and NLlac3, and NUlac4 and NLlac4) [Wang et al, 2006]. The resulting products were purified and used in DNA sequencing reactions with the BigDye TM terminator cycle sequencing chemistry (ver.…”