Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae.
Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae.
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映射减数分裂的单链DNA揭示了酿酒酵母中DNA双链断裂的新景观。
DOI：
10.1371/journal.pbio.0050324
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发表时间：
2007-12
期刊：
影响因子：
9.8
通讯作者：
Lichten, Michael
中科院分区：
文献类型：
--
作者：
Buhler, Cyril;Borde, Valerie;Lichten, Michael
Spo 11蛋白形成的DNA双链断裂（DSB）启动减数分裂重组。先前的DSB定位研究使用了断裂加工缺陷的rad 50 S或sae 2 Δ突变体来积累Spo 11连锁的DSB，并报告了由类似大小的“DSB冷”结构域分隔的大（≥ 50 kb）“DSB热”区域。在一些DSB冷区发生了大量的重组，表明DSB模式在rad 50 S或sae 2 Δ突变体中是不正常的。因此，我们开发了一种新的方法来定位全基因组，单链DNA（ssDNA）相关的DSB，这些DSB在有加工能力的，修复缺陷的dmc 1 Δ和dmc 1 Δ rad 51 Δ突变体中积累。在已知的热点观察到DSB，但也在大多数以前确定的“DSB冷”区域，包括着丝粒和端粒附近。尽管在rad 50 S突变体中大约40%的基因组是DSB-冷的，但来自dmc 1 Δ的减数分裂ssDNA的分析表明，这些区域中的大多数具有显著的DSB活性。在dmc 1 Δ、rad 50 S和野生型细胞中选定区域的DSB的Southern印迹分析证实了这些发现。因此，DSB的分布比以前认为的要均匀得多。比较dmc 1，dmc 1 rad 51和dmc 1 spo 11突变株中的DSB信号，将Dmc 1鉴定为全基因组的关键链交换活性，并证实了先前的结论，即spo 11诱导的病变启动所有减数分裂重组。在减数分裂过程中，存在于完整（二倍体）基因组中的每条染色体的两个拷贝聚集在一起，然后分离，形成单倍体配子（动物中的精子和卵子）。染色体之间交换DNA的交换对于染色体配对和分离至关重要，也促进了下一代的遗传多样性，为进化提供了原料。DNA双链断裂（DSB），这是由保守的Spo 11核酸酶形成，启动减数分裂重组。因此，DSB作图是用于确定减数分裂重组发生位置的标准遗传分析的替代方法。DSB在芽殖酵母突变体中被广泛定位，这些突变体不能从断裂末端去除Spo 11，从而阻止进一步的重组步骤。奇怪的是，这些研究表明，DSB是缺乏大区域的重组已知发生。我们开发了一种新的DSB作图方法，该方法纯化并分析了Spo 11去除后断裂处形成的单链DNA。这张新的图谱显示，DSB（以及由此推断的重组）实际上经常发生在几乎所有的芽殖酵母基因组中，其分布与重组在染色体配对和产生遗传多样性中的作用一致。这种新的作图方法将有助于研究其他生物的减数分裂重组和DNA损伤修复。作者开发了一种新的方法来检测全基因组DNA损伤，他们用它来表明减数分裂重组在芽殖酵母中的分布比以前认为的更均匀。
DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination. During meiosis, the two copies of each chromosome present in the full (diploid) genome come together and then separate, forming haploid gametes (sperm and eggs, in animals). Recombination, which swaps DNA between chromosomes, is critical for chromosome pairing and separation, and also promotes genetic diversity in the next generation, providing the feedstock for evolution. DNA double-strand breaks (DSBs), which are formed by the conserved Spo11 nuclease, initiate meiotic recombination. DSB mapping is thus an alternative to standard genetic analysis for determining where meiotic recombination occurs. DSBs have been most extensively mapped in budding yeast mutants that fail to remove Spo11 from break ends, blocking further recombination steps. Paradoxically, those studies indicated that DSBs are absent from large regions where recombination was known to occur. We developed a new DSB mapping method that purifies and analyzes the single-strand DNA formed at breaks after Spo11 removal. This new map shows that DSBs (and by inference, recombination) actually occur frequently throughout almost all of the budding yeast genome, in a distribution that is consistent with recombination's roles in chromosome pairing and in generating genetic diversity. This new mapping method will be useful for studying meiotic recombination and DNA damage repair in other organisms. The authors developed a new method to detect DNA damage genome-wide, and they used it to show that meiotic recombination is more uniformly distributed in budding yeast than was previously believed.