G-Quadruplex forming sequence motifs and genome instability

 描述(申请人提供)：突变和染色体重排是癌症基因组普遍存在的特征，与肿瘤发生相关的异常细胞活动有关。重组热点中，基因组重排以不一致的高频率启动，重复序列元素显著丰富，有可能折叠成非螺旋DNA二级结构。如果不加以解决，这种DNA结构可能会阻碍转录和复制，导致基因组不稳定。G-四链体或G4 DNA是一种四螺旋结构，由一连串的鸟嘌呤和一种对正常DNA交易构成挑战的DNA结构组成。然而，对分散在基因组中的含有鸟嘌呤序列的子集如何在遗传上变得不稳定的全面理解仍然缺乏。同样不清楚的是，这种潜在的基因组不稳定热点是如何得到控制的。申请者的长期研究目标是更好地了解整个基因组中自发突变和重排是如何以不同的速度发生的。这项拟议的研究的目的是表征在模式真核生物酿酒酵母中，是什么增强了与某些含有鸟嘌呤序列的序列相关的不稳定程度的显著升高。这一建议的中心假设是，G4基序的基因组不稳定性取决于基因组背景和调节含有鸟嘌呤序列的结构转换为G4 DNA结构的反式作用因子。这一假说建立在申请人产生的初步数据基础上，这些数据确定了在模型G4 DNA形成序列中调节不稳定性的两个关键因素-转录水平和拓扑异构酶I的活性。我们的具体目标是(1)确定拓扑异构酶I在G4 DNA相关基因组不稳定性中的作用(2)测试DNA损伤是否协同加剧G4形成序列的不稳定性，以及(3)在体内识别与共转录形成的G4 DNA特异结合的蛋白质。结合在体内检测G4DNA的分子方法，为定量测量G4基序以转录依赖方式发生的重组而开发的遗传分析将在AIM 1和2中用于确定Top1功能中断和DNA损伤积累对共转录形成的G4 DNA稳定性的影响。事实证明，遗传分析在生成相关的初步数据方面是有效和富有成效的。在AIM3下，通过染色质亲和纯化的方法将鉴定出可能在抑制G4DNA不稳定性方面发挥作用的新的反式激活因子。G4基序的稳定性与癌症生物学特别相关，因为在包含G4序列的基因组座位和在各种人类癌症中观察到的易位位置之间存在显着重叠。这项研究的结果最终可以加深我们对引发肿瘤发生重排的分子事件以及与治疗相关的继发性恶性肿瘤或耐药肿瘤发展的理解。

 DESCRIPTION (provided by applicant): Mutations and chromosome rearrangements are ubiquitous feature of cancer genomes and linked to the anomalous cellular activities associated with oncogenesis. Recombination hotspots, where genome rearrangements initiate at incongruently higher frequencies, are significantly enriched with repetitive sequence elements with the potential to fold into non-helical DNA secondary structures. When left unresolved, such DNA structures can impede transcription and replication leading to genome instability. G-quadruplex or G4 DNA is a tetra-helical structure consisting of runs of guanines and one of the DNA structures that presents a challenge to normal DNA transactions. Comprehensive understanding of how a subset of the guanine run- containing sequences interspersed in the genome becomes genetically unstable, however, is still lacking. Also not understood is how such potential hotspots of genome instability are kept under control. The long-term research goal of the applicant is to better understand how spontaneous mutagenesis and rearrangements occur at disparate rates throughout the genome. The goal of the proposed research is to characterize what potentiates the marked elevation in instability associated with certain guanine-run containing sequences in the model eukaryote, S. cerevisiae. The central hypothesis of this proposal is that genome instability at a G4 motif is contingent on both genomic context and trans-acting factors that modulate the structural transformation of a guanine-run containing sequence into G4 DNA structure. This hypothesis is founded on the preliminary data generated by the applicant that identified two critical factors in modulating instability at a model G4 DNA- forming sequence - the level of transcription and the activity of topoisomerase I. Our specific aims are designed to (1) define the role of topoisomerase I in the G4 DNA-associated genome instability (2) test whether DNA damage cooperatively aggravates the instability of G4 forming sequences and (3) Identify proteins binding specifically to co-transcriptionally formed G4 DNA in vivo. In combination with molecular approaches to detect G4 DNA in vivo, genetic assays developed for quantitative measurement of recombination occurring at a G4 motif in transcription-dependent manner will be employed in AIM 1 and 2 to determine the effect of disruption in Top1 function and accumulation of DNA damage on the stability of co- transcriptionally formed G4 DNA. The genetic assays have proved effective and productive in generating the relevant preliminary data. Under AIM3, novel trans-activating factors with possible roles in suppressing instability at the G4 DNA will be identified by chromatin affinity purification approach. The stability of G4 motifs is particularly relevant to th biology of cancer since there is a significant overlap between genomic loci containing G4 sequences and the sites of translocations observed in various human cancers. The result of the proposed study can ultimately enhance our understanding of the molecular events instigating the tumorigenic rearrangements as well as therapy-related secondary malignancy or drug-resistant tumor development.