Site-specific rates of excision repair of benzo[a]pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra.
Site-specific rates of excision repair of benzo[a]pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra.
复制标题
DOI：
10.1073/pnas.92.6.2204
复制
发表时间：
1995-03
影响因子：
11.1
通讯作者：
D. Wei;V. Maher;J. Mccormick
中科院分区：
文献类型：
--
作者：
D. Wei;V. Maher;J. Mccormick
当在 S 期早期用 (+/-)-7 β、8 α-二羟基-9 α、10 α-环氧-7,8,9,10-四氢苯并[a]芘 (BPDE) 处理修复能力良好的二倍体人成纤维细胞群时，正如次黄嘌呤磷酸核糖基转移酶基因 (HPRT) 正在复制一样，在 nt 212 处发现了 5% 的诱导碱基取代， 5% 的取代出现在外显子 3 的 nt 229 处。然而，当在 G1 早期对群体进行处理，以便在 S 期开始之前至少有 12 小时进行修复时，21% 的取代出现在 nt 212 处，10% 出现在 nt 229 处。在切除修复缺陷的细胞中，没有出现这种依赖于细胞周期的碱基取代分布差异。为了测试突变相对频率的增加是否是由于这些位点的低效修复所致，我们采用连接介导的 PCR 来测量 BPDE 加合物从 HPRT 基因外显子 3 的各个位点的去除率。在早期 G1 期用 0.5 µM BPDE 处理细胞，并立即或在 10、20 和 30 小时后收获以进行修复。分析外显子 3 的非转录链上加合物的原始分布和修复后剩余的分布，使用大肠杆菌 UvrABC 核酸酶切除加合物，将 5' 生物素化基因特异性引物与 DNA 退火，并用 Sequenase 2.0 延伸，在每次切割的位点生成平端。将接头连接至平端，并使用磁珠将所需片段与基因组 DNA 的其余部分分离，通过 PCR 扩增，并在测序凝胶上进行分析。特定长度片段的分布表明最初形成或保留在特定位点的 BPDE 加合物的相对数量。 HPRT 基因外显子 3 上各个位点的修复速率差异很大，并且在 nt 212 和 229 处非常慢，这有力地支持了低效 DNA 修复在突变热点形成中发挥重要作用的假设。
When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3. However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229. No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells. To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene. Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair. the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut. A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel. The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites. The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.