Eukaryotic Chromosome Replication

描述（由申请人提供）：真核DNA复制是通过在S期以有序序列在多个复制起点启动复制分叉来完成的。复制缺陷是导致包括癌症在内的各种人类疾病的基因组不稳定的主要来源；在酵母中，扰乱基因组的有序复制会导致基因组的不稳定。细胞已经进化出复杂的监控机制来监控其基因组的完整性和有序复制。因此，为了充分了解基因组不稳定性疾病的原因和机制，我们必须了解编排复制程序的分子机制以及面对DNA损伤和压力时确保忠实的基因组维护的机制。在研究酵母中已报道的一些染色体重排时，我们注意到，除了已知的重复元件（如trna和转座子）外，还有一些轶事证据表明复制的起源非常接近断点，这使我们进入了一条新的研究路线：询问起源是否本质上是基因组中不稳定的元件。通过对酿酒葡萄球菌与其基因组复制前的远亲瓦氏克卢维菌的进化比较，我们发现酿酒葡萄球菌的起源确实与基因组重排断点高度相关。因此，本提案有两个广泛的主题：7继续我们正在进行的理解起源作用和调控的分子机制的努力，特别关注起源如何应对复制应激和限制复制因子。7复制起点的存在和活性与基因组不稳定性之间的直接联系的实验测试。由于基因组重排是癌症进展的主要标志，我们认为这些研究将阐明起源功能和基因组稳定性之间的相互作用，并在长期内具有开发诊断和治疗策略的潜力。

DESCRIPTION (provided by applicant): Eukaryotic DNA replication is accomplished by initiating replication forks at multiple origins of replication in an ordered sequence during S phase. Replication defects are a major source of genomic instability leading to a variety of human disorders including cancer; in the yeast Saccharomyces cerevisiae, perturbing the orderly replication of the genome leads to genome instability. Cells have evolved elaborate surveillance mechanisms to monitor the integrity and orderly duplication of their genomes. To fully understand the causes and mechanisms of genome-instability disorders, therefore, it is crucial that we understand the molecular mechanisms choreographing the replication program and the mechanisms that ensure faithful genome maintenance in the face of DNA damage and stress. In examining some chromosomal rearrangements that have been reported in yeast, we note that in addition to the known repeated elements (such as tRNAs and transposons), there is anecdotal evidence for origins of replication very close to the breakpoint, leading us a to a new line of enquiry: to ask whether origins are intrinsically destabilizing elements in the genome. Drawing on evolutionary comparisons of S. cerevisiae with its distant, pre-genome- duplication relative Kluyveromyces waltii, we have found that origins in S. cerevisiae are indeed very highly correlated with genome rearrangement breakpoints. This proposal therefore has two broad themes: 7 A continuation of our ongoing efforts to understand the molecular mechanisms of origin action and regulation, with particular focus on how origins respond to replication stress and limiting replication factors. 7 Experimental tests for a direct link between the presence and activity of replication origins and genome instability. Because genome rearrangements are a major hallmark of cancer progression, we feel that these lines of enquiry will elucidate the interplay between origin function and genome stability, and in the long term have potential for developing both diagnostic and treatment strategies. PUBLIC HEALTH RELEVANCE: Perturbations in the orderly duplication of chromosomal DNA are a major source of genomic instability leading to a variety of human disorders including cancer. At the same time, there is evidence that origins of replication, the sites where DNA synthesis is initiated, are themselves potential agents of genome rearrangements. In this work, we propose to continue our investigations into the mechanisms that modulate the orderly progression of replication, and begin exploring the possible role of replication origins in leading to genome instability. This work will expand our understanding of the mechanisms underlying genome disorders, shed light on questions of health importance, and contribute to our ongoing efforts to understand the interplay between genome structure and replication.