Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, United States.
Fungal Genet Biol. 2013 Jan;50:33-43. doi: 10.1016/j.fgb.2012.10.007. Epub 2012 Nov 17.
Double-stranded break (DSB) repair during meiotic recombination in yeast Saccharomyces cerevisiae leads to the formation of heteroduplex DNA, a hybrid DNA molecule composed of single strands from two homologous chromosomes. Differences in sequence between the strands within heteroduplex DNA generate mismatches or large unpaired loops that are substrates for repair. At least two pathways function to repair large loops that form within heteroduplex DNA: the RAD1-dependent large loop repair (LLR) pathway and another as yet uncharacterized RAD1-independent LLR pathway. Repair of large loops during meiotic recombination is especially important for the genomic stability of the repetitive DNA sequences known as minisatellites. Minisatellite DNA tracts are generally stable during mitotic cell divisions but frequently alter in length during meiosis. Using a yeast minisatellite system in which the human minisatellite associated with the HRAS1 proto-oncogene has been inserted into the recombination hotspot region upstream of HIS4 in S. cerevisiae, our lab previously showed that the RAD1-dependent LLR pathway controls minisatellite length expansions, but not contractions. Here we show that minisatellite length expansions are controlled by the products of the CSM3 and TOF1 genes, while contractions are controlled by MRC1. By examining meiotic segregation patterns in yeast strains heterozygous for the 26bp his4-lopd insert, we found that deleting CSM3 caused a loss of LLR activity similar to that seen in a RAD1 mutant. Double mutant analysis revealed that failure to repair loops is exacerbated upon deleting both RAD1 and CSM3 - specifically the type of repair that fills in loops, which would generate minisatellite length expansions. A model for minisatellite length alteration based on these results is presented.
在酵母酿酒酵母的减数分裂重组过程中，双链断裂 (DSB) 修复会导致异源双链 DNA 的形成，这是一种由两个同源染色体的单链组成的杂交 DNA 分子。异源双链 DNA 内链之间的序列差异会产生错配或未配对的大环，这些都是修复的底物。至少有两种途径可用于修复异源双链 DNA 内形成的大环：RAD1 依赖性大环修复 (LLR) 途径和另一种尚未被描述的 RAD1 非依赖性 LLR 途径。在减数分裂重组过程中修复大环对于称为卫星 DNA 的重复 DNA 序列的基因组稳定性尤为重要。卫星 DNA 序列在有丝分裂细胞分裂过程中通常是稳定的，但在减数分裂过程中长度经常会发生改变。在使用酵母卫星 DNA 系统中，我们的实验室之前已经将与 HRAS1 原癌基因相关的人类卫星 DNA 插入到酿酒酵母 HIS4 上游的重组热点区域中，发现 RAD1 依赖性 LLR 途径控制着卫星 DNA 的长度扩展，但不控制收缩。在这里，我们表明卫星 DNA 的长度扩展受 CSM3 和 TOF1 基因产物的控制，而收缩则受 MRC1 的控制。通过检查杂合了 26bp his4-lopd 插入的酵母菌株的减数分裂分离模式，我们发现删除 CSM3 会导致 LLR 活性的丧失，类似于 RAD1 突变体的情况。双突变体分析表明，当同时删除 RAD1 和 CSM3 时，无法修复环会加剧，特别是会填补环的修复类型，这会导致卫星 DNA 的长度扩展。基于这些结果提出了一种卫星 DNA 长度改变的模型。
