G-Quadruplex forming sequence motifs and genome instability

 描述（由申请人提供）：突变和染色体重排是癌症基因组的普遍特征，与肿瘤发生相关的异常细胞活动有关。其中基因组重排以不一致的更高频率开始的扩增热点显著富含具有折叠成非螺旋DNA二级结构的潜力的重复序列元件。当不解决时，这样的DNA结构可以阻碍转录和复制，导致基因组不稳定。G-quadruplex或G4 DNA是一种由鸟嘌呤组成的四螺旋结构，是对正常DNA处理提出挑战的DNA结构之一。然而，对于散布在基因组中的含鸟嘌呤序列的子集如何变得遗传不稳定的全面理解仍然缺乏。同样不清楚的是，这些潜在的基因组不稳定热点是如何被控制的。申请人的长期研究目标是更好地理解自发诱变和重排如何在整个基因组中以不同的速率发生。这项研究的目的是为了确定是什么因素导致了真核生物模型S。啤酒。该提议的中心假设是，基因组在G4基序的不稳定性取决于基因组背景和调节含鸟嘌呤序列向G4 DNA结构的结构转化的反式作用因子。该假设建立在申请人产生的初步数据上，该数据鉴定了在模型G4 DNA形成序列处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损伤是否协同地破坏G4形成序列的不稳定性;（3）鉴定体内特异性结合共转录形成的G4 DNA的蛋白质。结合体内检测G4 DNA的分子方法，将在AIM 1和2中采用为定量测量以转录依赖性方式在G4基序处发生的重组而开发的遗传测定，以确定Top1功能破坏和DNA损伤累积对共转录形成的G4 DNA稳定性的影响。遗传分析在产生相关初步数据方面证明是有效和富有成效的。在AIM 3下，将通过染色质亲和纯化方法鉴定可能在抑制G4 DNA不稳定性中起作用的新型反式激活因子。G4基序的稳定性与癌症的生物学特别相关，因为含有G4序列的基因组基因座与在各种人类癌症中观察到的易位位点之间存在显著重叠。拟议的研究结果可以最终提高我们的理解的分子事件煽动肿瘤重排以及治疗相关的继发性恶性肿瘤或耐药肿瘤的发展。

 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.