Ohta K, Shibata T, Nicolas A
Laboratory of Cellular and Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
EMBO J. 1994 Dec 1;13(23):5754-63. doi: 10.1002/j.1460-2075.1994.tb06913.x.
Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination.
在酿酒酵母减数分裂过程中，瞬时双链断裂（DSB）发生在重组热点区域，并且被认为启动了染色体间大部分（如果不是全部）的同源重组。为了揭示DSB形成过程中活跃的调控机制，我们监测了减数分裂过程中ARG4和CYS3重组热点区域局部染色质结构的变化。对分离出的减数分裂染色质进行微球菌核酸酶（MNase）消化，然后进行间接末端标记，结果显示两个位点的DSB位点对MNase高度敏感，并且在减数分裂DSB和重组产物出现之前，它们的敏感性增加了2至4倍。其他敏感位点没有明显变化。对ARG4位点高重组和低重组构建体的研究还表明，DSB位点的MNase敏感性与DSB的程度和基因转换率相关。这些结果表明，减数分裂早期的局部染色质结构及其修饰在减数分裂DSB的定位和频率中起重要作用，从而导致减数分裂重组。
