Topoisomerase 1 and mutagenesis in yeast

描述(申请人提供)：转录对萌芽酵母的诱变有很强的刺激作用，大多数与转录相关的突变都是短的缺失，移除了2-5个碱基串联重复序列的一个拷贝。这些签名缺失需要Top1的活性，Top1是一种拓扑异构酶，它通过划破一条DNA链，将完整的DNA链穿过缺口，然后重新密封缺口，从而移除与转录相关的超级螺旋。类似的短缺失签名与错误结合到DNA中的核糖核苷酸(RNMP)移除失败有关，该签名同样依赖于Top1的活性。尽管依赖转录和依赖rNMP的缺失累积的位置存在重叠，但各自的热点并不完全一致。因此，我们认为有两种不同类型的Top1产生的损伤导致缺失：(1)Top1裂解复合体(Top1cc)在正常的切割-连接循环中捕获到DNA上；(2)当Top1在rNMP处裂解时产生不可逆的缺口。然而，这两种类型的损伤都被建议处理成位于相关串联重复序列内的小间隙。随后互补链上重复序列之间的错位将使末端结合在一起，从而促进连接。拟议的研究将表征两种类型的缺失热点中每一种类型的Top1依赖的突变机制。在目标1中，我们将在体外绘制Top1切割位点的图谱，然后使用这些信息来确定切割位点和体内发生缺失的串联重复之间的位置关系。Aim 2将使用候选基因方法来识别将Top1产生的损伤处理成小间隙的蛋白质。目标3将集中于依赖rNMP的热点；将鉴定插入rNMP的DNA聚合酶，并将检查细胞周期阶段与突变的相关性。最后，目标4将定义在转录过程中捕获Top1cc的因素，并将探索喜树碱的突变潜力，喜树碱是一种稳定Top1cc的化疗药物。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的基因组稳定性问题相关。 与公共卫生相关：突变为进化过程提供了原材料，并导致了许多人类疾病，特别是癌症。拟议的实验将使用萌芽酵母作为模型系统，以探索特定来自Top1活性的突变。Top1是一种拓扑异构酶，可解析与转录相关的超级线圈。这些研究将探索Top1依赖的诱变的一般机制以及喜树碱的诱变潜力，喜树碱是一种针对Top1的化疗药物。

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. PUBLIC HEALTH RELEVANCE: Mutations provide the raw material for evolutionary processes and are causative in a number of human diseases, especially cancer. The proposed experiments will use budding yeast as a model system to explore mutagenesis that derives specifically from the activity of Top1, a topoisomerase that resolves the supercoils associated with transcription. These studies will explore the general mechanism of Top1-dependent mutagenesis as well as the mutagenic potential of camptothecin, a chemotherapeutic drug that specifically targets Top1.