Topoisomerase 1 and mutagenesis in yeast

描述（由申请人提供）：转录对出芽酵母的诱变有很强的刺激作用，大多数转录相关的突变是短缺失，删除一个2-5 bp串联重复的拷贝。这些特征缺失需要Top1的活性，Top1是一种拓扑异构酶，它通过切割一条DNA链，将完整的DNA链穿过缺口，然后重新密封缺口，从而去除转录相关的超级线圈。类似的短缺失特征与无法去除错误结合到DNA中的核糖核苷酸（rNMPs）有关，这种特征同样依赖于Top1的活性。尽管转录依赖性缺失和rnmp依赖性缺失的累积存在重叠，但各自的热点并不完全一致。因此，我们提出有两种不同类型的Top1产生的损伤导致缺失：(1)在正常的切割-连接周期中被捕获在DNA上的Top1切割复合体（Top1cc）和(2)当Top1在rNMP上切割时产生的不可逆缺口。然而，这两种类型的病变都被建议处理成一个小的间隙，该间隙位于相关的串联重复序列中。随后，互补链重复序列之间的不对齐将使末端连接在一起，从而促进结扎。所提出的研究将描述top1依赖性突变在两种缺失热点上的机制。在Aim 1中，我们将绘制体外Top1切割位点，然后使用这些信息来定义切割位点与体内发生缺失的串联重复序列之间的位置关系。Aim 2将使用候选基因方法来鉴定将top1产生的病变加工成小间隙的蛋白质。目标3将侧重于依赖rNMP的热点；将鉴定插入rNMPs的DNA聚合酶，并检查细胞周期阶段与突变的相关性。最后，Aim 4将定义在转录过程中捕获Top1cc的因子，并探索喜树碱（一种稳定Top1cc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相关。

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.