Genetic regulation of genome stability in yeast

描述(申请人提供)：所有生物的基因组都会发生变化，包括染色体数量、染色体结构(易位、缺失、重复)或单碱基对突变(点突变)。虽然这些变化在野生型细胞中的频率很低，但某些突变(突变子)极大地提高了不稳定性的速度。大多数实体肿瘤的染色体重组率都很高。在这项提案中，DNA微阵列和高通量DNA测序将被用于对遗传不稳定的酿酒酵母菌株的重组事件进行全基因组分析，以此作为理解具有高水平染色体重排的癌症的模型。此外，缺乏拓扑异构酶的酵母菌株中重组事件的图谱将与理解拓扑异构酶抑制剂用于化疗的可能后果有关。这种分析的一个基本原理是，有丝分裂重组水平高的区域也有高水平的双链DNA断裂(DSB)，而映射重组事件的位置就映射了这些DSB的位置。在两条同源染色体不完全相同的二倍体品系中，有丝分裂重组事件会导致杂合性丢失(LOH)。构建了约55,000个单核苷酸多态(SNPs)杂合的二倍体菌株，并将使用可以检测这些多态的菌株是杂合还是纯合的微阵列(SNP阵列)来定位LOH事件。这些事件将在已知具有非常高遗传不稳定性的菌株中被定位在整个基因组中，包括：1)低水平的αDNA聚合酶的菌株，2)tel1 Mec1 sml1菌株(缺乏人类ATM和ATR基因的同源)，以及3)拓扑异构酶Top1p和/或Top2p突变的菌株。这些研究将确定在这三种不同类型的基因组不稳定条件下容易形成DSB的基因组区域。作为原则的证明，我们绘制了200多个LOH事件，这些事件发生在αDNA聚合酶水平降低10倍的菌株中，并表明这些事件与已知的减慢DNA复制分叉的DNA序列基序非随机相关。为了补充这一方法，将使用微阵列来定位具有低αDNA聚合酶或拓扑异构酶基因突变的菌株中的伽马-H_2AX的位置。由于伽马-H_2AX被招募到DNA损伤的部位，因此经常与LOH相关的区域可能与高水平的伽马-H_2AX的区域共定位。还将对上述相同类型的三种菌株的分离株进行高通量DNA测序，以检测SNP微阵列无法检测到的遗传变化(单碱基对变化和小插入/缺失)。最后，我们计划在暴露于拓扑异构酶抑制剂的哺乳动物细胞中定位LOH事件。

DESCRIPTION (provided by applicant): The genomes of all organisms are subject to change including alterations in chromosome number, chromosome structure (translocations, deletions, duplications), or single-base-pair mutations (point mutations). Although the frequency of these changes is very low in wild-type cells, certain mutations (mutators) greatly elevate the rate of instability. Most solid tumors have very high rates of chromosome rearrangements. In this proposal, DNA microarrays and high-throughput DNA sequencing will be used to perform a genome-wide analysis of recombination events in strains of the yeast Saccharomyces cerevisae that are genetically unstable as a model for understanding cancers with high levels of chromosome rearrangements. In addition, the mapping of recombination events in yeast strains lacking topoisomerases will be relevant to understanding the possible consequences of the chemotherapeutic use of topoisomerase inhibitors. One rationale for this analysis is that regions that have high levels of mitotic recombination also have high levels of double-stranded DNA breaks (DSBs), and that mapping the positions of the recombination events maps the positions of these DSBs. Mitotic recombination events in diploid strains in which the two homologous chromosomes are not identical result in loss of heterozygosity (LOH). Diploid strains heterozygous for about 55,000 single-nucleotide-polymorphisms (SNPs) were constructed and will be analyzed using microarrays that can detect whether strains are heterozygous or homozygous for these polymorphisms (SNP arrays) to map LOH events. These events will be mapped throughout the genome in strains known to have very high levels of genetic instability including: 1) strains with low levels of alpha DNA polymerase, 2) tel1 mec1 sml1 strains (lacking homologues of the human ATM and ATR genes), and 3) strains with mutations in the topoisomerases Top1p and/or Top2p. These studies will define regions of the genome that are prone to DSB formation under these three different types of genome-destabilizing conditions. As a proof of principle, we mapped more than 200 LOH events in strains with 10-fold reduced levels of alpha DNA polymerase and showed that these events are non-randomly associated with DNA sequence motifs known to slow DNA replication forks. To complement this approach, microarrays will be used to map the locations of gamma-H2AX in strains with low alpha DNA polymerase or mutations in topoisomerase genes. Since gamma-H2AX is recruited to sites of DNA damage, the regions frequently associated with LOH are likely to co-localize with regions that have high levels of gamma-H2AX. High-throughput DNA sequencing of isolates of the same three types of strains described above will also be done to detect genetic alterations (single-base-pair changes and small insertions/deletions) that cannot be detected by SNP microarrays. Finally, we plan to map LOH events in mammalian cells that are exposed to topoisomerase inhibitors.