Topoisomerase 1 and mutagenesis in yeast

描述（由申请人提供）：转录对芽殖酵母中的诱变具有强烈的刺激作用，并且大多数转录相关突变是短缺失，其去除了2-5 bp串联重复序列的一个拷贝。这些特征缺失需要Top1的活性，Top1是一种拓扑异构酶，通过切割一条DNA链，使完整的链通过切口，然后重新密封切口来去除转录相关的超螺旋。一个类似的短缺失特征与去除错误掺入DNA中的核糖核苷酸（rNMP）的失败有关，并且该特征同样取决于Top1活性。尽管转录依赖性缺失和rNMP依赖性缺失累积之间存在重叠，但各自的热点并不完全重合。因此，我们提出，有两种不同类型的Top1产生的病变，导致删除：（1）Top1切割复合物（Top1 cc），成为捕获的DNA在正常的切割连接周期和（2）不可逆的切口时，Top1切割rNMP创建。然而，这两种类型的病变，建议被处理成一个小的差距，驻留在相关的串联重复。互补链上重复序列之间的后续错位将使末端在一起以促进连接。拟议的研究将表征在两种类型的缺失热点中的每一种的Top1依赖性诱变的机制。在目标1中，我们将在体外映射Top1切割位点，然后使用这些信息来定义切割位点和串联重复序列之间的位置关系，其中在体内发生缺失。目标2将使用候选基因方法来鉴定将Top1产生的病变处理成小间隙的蛋白质。目标3将集中于rNMP依赖性热点;将鉴定插入rNMP的DNA聚合酶，并将检查细胞周期阶段与诱变的相关性。最后，目标4将定义在转录过程中捕获Top1 cc的因素，并将探索喜树碱（一种稳定Top1 cc的化疗药物）的致突变潜力。鉴于DNA结构和基本DNA代谢过程的普遍性，酵母系统的结果将与高等真核生物基因组稳定性问题有关。

DESCRIPTION (provided by applicant): Transcription has a strong stimulatory effect on mutagenesis in budding yeast, and most transcription-associated mutations are short deletions that remove one copy of a 2-5 bp tandem repeat. These signature deletions require the activity of Top1, a topoisomerase that removes transcription-associated supercoils by nicking one strand of DNA, passing the intact strand through the nick, and then resealing the nick. A similar, short-deletion signature has been associated with the failure to remove ribonucleotides (rNMPs) misincorporated into DNA, and this signature likewise depends on Top1 activity. Though there is overlap between where transcription-dependent and rNMP-dependent deletions accumulate, the respective hotspots are not entirely coincident. We thus propose that there are two distinct types of Top1-generated lesions that lead to deletions: (1) a Top1 cleavage complex (Top1cc) that becomes trapped on DNA during the normal cleavage-ligation cycle and (2) an irreversible nick created when Top1 cleaves at an rNMP. Both types of lesions, however, are proposed to be processed into a small gap that resides within the relevant tandem repeat. Subsequent misalignment between repeats on the complementary strands will bring the ends together to facilitate ligation. The proposed studies will characterize the mechanisms of Top1-dependent mutagenesis at each of the two types of deletion hotspots. In Aim 1, we will map Top1 cleavage sites in vitro, and then use this information to define the positional relationship between the cleavage site and the tandem repeat where deletions occur in vivo. Aim 2 will use a candidate gene approach to identify proteins that process Top1-generated lesions into a small gap. Aim 3 will focus on rNMP- dependent hotspots; the DNA polymerase that inserts rNMPs will be identified and the relevance of cell-cycle phase to mutagenesis will be examined. Finally, Aim 4 will define factors that trap the Top1cc during transcription and will explore the mutagenic potential of camptothecin, a chemotherapeutic drug that stabilizes the Top1cc. Given the universality of DNA structure and basic DNA metabolic processes, results in the yeast system will be relevant to issues of genome stability in higher eukaryotes.