MOLECULAR MECHANISMS OF DNA REPAIR IN YEAST

酵母有丝分裂细胞中DNA修复系统的鉴定 酿酒酵母正在检查a）它们对经历 减数分裂，和B）相应基因在正常减数分裂中的作用。 我们 已经开发出独特的蔗糖梯度技术来检查修复后， 低剂量的紫外线或电离辐射，并遵循减数分裂DNA的变化 在减数分裂期间。 RAD50、RAD52和RAD57基因在DNA修复中是必需的 有丝分裂细胞中的双链断裂。 它们也需要用于 减数分裂 这些基因的突变破坏了正常的减数分裂重组， 似乎RAD 50在减数分裂的早期步骤起作用。 稀有单链 在rad52和rad57菌株中观察到中断（SSI）， 减数分裂DNA合成的开始，这些似乎与 重组 温和的隔离技术使得 SSIs作为DNA断裂的特征;许多具有3 'OH和5' PO4 特米尼 SSI似乎不是随机分布的，基于 涉及特定染色体区域探针的实验，表明 涉及正常减数分裂重组的特定位点或区域。 虽然rad52和rad57突变体在减数分裂重组中有缺陷， 重组体可以在进行还原分裂之前回收。 rad52突变体中的频率远低于RAD+菌株; 在RAD 57突变体中频率稍低。 重组体还 与RAD+中的有质的不同。 当减数分裂停止， rad52或rad57暴露于生长培养基，重组体不 恢复 这是由于重组体需要延长的时间， 形式，并表明rad52和rad57在中间步骤被阻断 在重组中。 重组中间体被缓慢地分解， 拆分前的生长会阻止重组体的出现。

DNA repair systems identified in mitotic cells of the yeast Saccharomyces cerevisiae are being examined for a) their protection of cells undergoing meiosis, and b) the role of the corresponding genes in normal meiosis. We have developed unique sucrose gradient techniques to examine repair after low doses of UV or ionizing radiation and to follow changes in meiotic DNA during meiosis. The RAD50, RAD52 and RAD57 genes are essential in the repair of DNA double-strand breaks in mitotic cells. They are also required for meiosis. Mutations in these genes abolish normal meiotic recombination; it appears that RAD50 acts at an early step in meiosis. Rare single-strand interruptions (SSIs) were observed in rad52 and rad57 strains shortly after the beginning of meiotic DNA synthesis and these appear to be related to recombination. Gentle isolation techniques have allowed the characterization of SSIs as breaks in DNA; many have 3' OH and 5' PO4 termini. The SSIs do not appear to be randomly distributed, based on experiments involving probes for specific chromosomal regions, suggesting specific sites or regions involved in normal meiotic recombination. While rad52 and rad57 mutants are defective in meiotic recombination, recombinants can be recovered prior to commitment to reductional division. The frequency in rad52 mutants is much lower than in RAD+ strains; the frequency is somewhat lower in rad57 mutants. The recombinants are also qualitatively different from those in RAD+. When meiosis is arrested and rad52 or rad57 are exposed to growth medium, recombinants are not recovered. This is due to the extended time necessary for recombinants to form, and suggests that rad52 and rad57 are blocked at an intermediate step in recombination. The recombination intermediates are resolved slowly, and growth prior to resolution prevents the appearance of recombinants.