Quantitative Genome-Wide Measurements of Meiotic DNA Double-Strand Breaks and Protein Binding in S. pombe.

Quantitative Genome-Wide Measurements of Meiotic DNA Double-Strand Breaks and Protein Binding in S. pombe.
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粟酒裂殖酵母减数分裂 DNA 双链断裂和蛋白质结合的全基因组定量测量。

DOI:
10.1007/978-1-4939-6340-9_2
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发表时间:
2017
期刊:
Methods in molecular biology (Clifton, N.J.)
影响因子:
--
通讯作者:
Smith,GeraldR
Smith,GeraldR
中科院分区:
--
文献类型:
--
作者:
Hyppa,RandyW;Fowler,KyleR;Smith,GeraldR

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裂殖酵母尤其适合于研究减数分裂的分子细节.实验用S.在不同温度下经历几乎同步减数分裂的粟酒例如,研究集中在启动减数分裂重组的程序性DNA双链断裂(DSB)已被证明是非常有益的。在减数分裂中,DNA的某些区域比周围区域具有更频繁的DSB。这些DSB热点可以通过限制性片段的Southern印迹杂交来可视化,所述限制性片段的大小范围从DNA酶(kb)到兆碱基(Mb)。最近,全基因组分析的好处,以地图的减数分裂DSB的分布和频率已经实现,分辨率下降到核苷酸水平。已经观察到以前无法检测到的罕见的非热点DSB,从而更好地理解重组是如何调节的。另外的全基因组分析已经显示了特异性结合DSB热点的蛋白质,提供了对DSB起始复合物如何发挥功能的深入了解。我们在这里描述实现这些结果的详细方法。
The fission yeastSchizosaccharomyces pombeis especially well suited for studying meiosis in molecular detail. Experiments withS. pombestrains that undergo a nearly synchronous meiosis—at variable temperatures—have elucidated the mechanisms of meiotic progression and the proteins that are involved. For example, studies focused on the initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs) have proven exceptionally informative. In meiosis, some regions of DNA have more frequent DSBs than the surrounding regions. These DSB hotspots can be visualized by Southern blot hybridization of restriction fragments ranging from kilobases (kb) to megabases (Mb) in size. More recently, the benefits of genome-wide analysis to map the distribution and frequency of meiotic DSBs have been attained, with resolution down to the nucleotide level. Infrequent, non-hotspot DSBs previously not detectable have been observed, creating a better understanding of how recombination is regulated. Additional genome-wide analyses have shown proteins that bind specifically to DSB hotspots, providing insight into how the DSB initiation complex functions. We describe here detailed methods for achieving these results.
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