Genetic regulation of genome stability in yeast

描述（由申请人提供）：所有生物体的基因组均会发生变化，包括染色体数目、染色体结构（易位、缺失、重复）或单碱基对突变（点突变）的改变。虽然这些变化的频率在野生型细胞中非常低，但某些突变（增变因子）大大提高了不稳定性的速率。大多数实体瘤具有非常高的染色体重排率。在这项提议中，DNA微阵列和高通量DNA测序将用于对遗传不稳定的酿酒酵母菌株中的重组事件进行全基因组分析，作为了解具有高水平染色体重排的癌症的模型。此外，在缺乏拓扑异构酶的酵母菌株中的重组事件的映射将与理解拓扑异构酶抑制剂的化疗用途的可能后果有关。 这种分析的一个基本原理是，具有高水平有丝分裂重组的区域也具有高水平的双链DNA断裂（DSB），并且绘制重组事件的位置映射这些DSB的位置。二倍体菌株中两条同源染色体不相同的有丝分裂重组事件导致杂合性丢失（洛）。构建了约55，000个单核苷酸多态性（SNP）杂合的二倍体菌株，并将使用可以检测菌株对于这些多态性是杂合还是纯合的微阵列（SNP阵列）进行分析，以绘制洛高事件。这些事件将在已知具有非常高水平遗传不稳定性的菌株的整个基因组中进行定位，包括：1）具有低水平α DNA聚合酶的菌株，2）tel 1 mec 1 sml 1菌株（缺乏人ATM和ATR基因的同源物），和3）具有拓扑异构酶Top1 p和/或Top2 p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易于形成DSB的基因组区域。作为原理的证明，我们在α DNA聚合酶水平降低10倍的菌株中绘制了200多个洛缺失事件，并表明这些事件与已知减缓DNA复制叉的DNA序列基序非随机相关。为了补充这种方法，微阵列将被用来映射的位置γ-H2 AX在菌株与低α DNA聚合酶或突变的拓扑异构酶基因。由于γ-H2 AX被募集到DNA损伤的位点，所以经常与洛缺失相关的区域可能与具有高水平γ-H2 AX的区域共定位。还将对上述相同三种类型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异（单碱基对变化和小插入/缺失）。最后，我们计划映射洛事件在哺乳动物细胞暴露于拓扑异构酶抑制剂。 公共卫生相关性：在大多数实体瘤细胞中发现遗传不稳定性，导致高水平的染色体重排。我们将使用DNA微阵列和高通量DNA测序来对具有非常高水平的染色体重排的酿酒酵母菌株进行全基因组分析。我们对容易重排的酵母染色体区域的定位将有助于我们了解与癌症相关的遗传不稳定性。

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. PUBLIC HEALTH RELEVANCE: Genetic instability, resulting in high levels of chromosome rearrangements, is found in the cells of most solid tumors. We will use DNA microarrays and high-throughput DNA sequencing to perform a genome-wide analysis of strains of the yeast Saccharomyces cerevisae that have very high levels of chromosome rearrangements. Our mapping of yeast chromosome regions that are prone to rearrangements will help us understand the genetic instability associated with cancer.