High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.
High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.
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DOI：
10.1371/journal.pgen.1005938
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发表时间：
2016-03
期刊：
影响因子：
4.5
通讯作者：
Petes TD
中科院分区：
文献类型：
--
作者：
Andersen SL;Zhang A;Dominska M;Moriel-Carretero M;Herrera-Moyano E;Aguilera A;Petes TD
酿酒酵母RAD 3基因是人XPD的同源物，XPD是编码TFIIH复合物的DNA解旋酶的必需基因，其参与核苷酸切除修复（NER）和转录。RAD 3的一些突变等位基因（rad 3 -101和rad 3 -102）在DNA修复中具有部分缺陷和强超重组（hyper-Rec）表型。先前的研究表明，与rad 3 -101和rad 3 -102相关的hyper-Rec表型可以解释为通过复制转化为重组双链断裂（DSB）的持续单链DNA缺口的结果。以前用于表征rad 3菌株的hyper-Rec表型的系统未检测到有丝分裂重组的相互产物。我们进一步的特点，这些事件使用一个系统，其中有丝分裂重组的相互产品被回收。rad 3 -101和rad 3 -102都将相互交叉的频率提高了约100倍。这些事件的映射显示，这些交叉的四分之三反映了在两个姐妹染色单体的相同位置形成的DSB（双姐妹染色单体断裂，DSCBs）。其余部分反映了在单个染色单体中形成的DSB（单个染色单体断裂，SCB）。DSCB与SCB的比率类似于在野生型细胞中观察到的自发重组事件。我们绘制了整个基因组中的216个突变，包括交叉、基因转换、缺失和重复。我们发现这些重组事件的位置与γ-H2 AX升高的区域之间存在显着关联。此外，在第XV号染色体左端附近的IMA 2和HXT 11基因处存在缺失和重复的热点。这些数据与我们以前的分析自发有丝分裂重组事件的比较表明，在野生型细胞中的一个子集的自发事件可能是由不完全的NER反应，而DSCBs，不能修复的姐妹染色单体重组，同源染色体之间的有丝分裂重组的一个主要来源。二倍体真核生物中每对同源染色体的两个成员通常是杂合的许多单核苷酸多态性（SNP）。同源染色体之间的有丝分裂重组可导致这些SNP的杂合性丢失。虽然有丝分裂重组作为遗传多样性的来源是有益的，但杂合子个体中肿瘤抑制基因的野生型等位基因的丢失是致癌的重要因素。在目前的研究中，我们映射重组事件在酵母菌株具有突变的RAD 3，导致一个强大的超重组表型。RAD 3（人类XPD基因的同源物）在转录和核苷酸切除修复中起作用。我们发现rad 3突变体在整个酵母基因组中的交叉率提高了100倍，并且这些事件与酵母基因组中脆弱的区域非随机相关，即使在野生型细胞中也是如此。我们还表明，大多数同源染色体之间的重组事件是在同一位置被打破的两个姐妹染色单体的修复的结果，类似于在野生型细胞中发生的事件类型。
The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes. The two members of each pair of homologous chromosomes in diploid eukaryotes are usually heterozygous for many single-nucleotide polymorphisms (SNPs). Mitotic recombination between homologous chromosomes can lead to loss of heterozygosity of these SNPs. Although mitotic recombination can be beneficial as a source of genetic diversity, loss of wild-type alleles of tumor suppressor genes in heterozygous individuals is an important contributor to carcinogenesis. In the current study, we map recombination events in yeast strains that have mutations in RAD3 that result in a strong hyper-recombination phenotype. RAD3 (a homolog of the essential human XPD gene) has roles in both transcription and nucleotide excision repair. We find that the rad3 mutants have a 100-fold elevated rate of crossovers throughout the yeast genome, and these events are non-randomly associated with regions of the yeast genome that are fragile, even in wild-type cells. We also show that most of the recombination events between homologous chromosomes are a consequence of the repair of two sister chromatids that are broken at the same position, similar to the types of events that occur in wild-type cells.