Dynamic functions of DNA2 counteract DNA replication stresses and tumorigenesis

总结 癌细胞的产生和发展是由于遗传和表观遗传改变的积累， 癌症表型这些改变的一个潜在原因是“难以复制”的DNA序列 (DTR)并充当复制应激的内源性来源。在整个基因组中有两大类 相互重叠的DTR序列，包括着丝粒区域的小卫星和微卫星 和端粒中的G-四链体（G4 s）。在DTR中形成的这种二级结构，如果没有适当地解析， 可能会阻碍DNA复制叉的移动，导致基因组不稳定。然而，细胞已经发展出 解决这些障碍的机制，以实现高效和忠实的DNA复制。最著名的方法是 解析G4和其他二级结构是通过DNA解旋酶解旋结构。在过去 在资助期间，我们阐明了核酸酶/解旋酶DNA 2促进DTR序列处的DNA复制。在 我们的初步研究表明，DNA错配修复蛋白MSH 2，MutSα的一个组成部分， 复合物，结合G4和DNA 2，并强烈刺激DNA 2切割G4结构。辐射， DNA 2或MSH 2的消除或组蛋白H1 c泛素化的缺乏导致G4积累。所以我们 假设：1）DNA 2与MutSα复合，切除并修复G4结构，以促进DNA复制 2）泛素化的H1 c将DNA 2/MutSα复合物募集到携带G4的DNA末端， 用于DSB的同源定向DNA修复（HDR）的链断裂（DSB）;和3）损害G4的基因突变 解析过程使个体对诱导或稳定G4结构的化学物质敏感，导致基因组 重排和癌症起始。我们建议定义G4切除的重要分子方面 在DNA复制或DSB修复过程中。因为G4是染色体结构元件， 和调节基因表达的表观遗传基序，G4切除必须严格控制。重要的是要 阐明DNA 2/MutSα是如何被募集到G4结构进行切除修复的。我们将定义 泛素E3连接酶ITCH或RNF 8介导的H1 c泛素化如何诱导DNA 2/MutSα切割G4 DNA复制和DSB修复过程中的结构。此外，大量的环境污染 化合物（ECCs）可以特异性结合G4结构并改变G4分解的动力学。我们预计 一旦基因突变损害了G4分解途径，G4稳定剂可以协同作用， 复制应激、DSB和基因组重排。因此，我们将确定是否结合基因和 抑制适当的G4分解/修复的环境因素在促进基因组不稳定性方面显示出协同作用， 癌症起始。我们的综合分析将确定G4切除在S期的途径， 通过HDR进行DNA DSB修复，并将提供G4稳定ECC降低癌症阈值的证据， 特别是对于在DNA 2介导的G4和其它二级结构解析途径中具有缺陷的个体。

SUMMARY Cancer cells arise and progress due to accumulation of genetic and epigenetic alterations that contribute to cancer phenotypes. One potential cause of these alterations are DNA sequences that are “difficult-to-replicate” (DTR) and act as an endogenous source of replication stress. Across the genome there are two major classes of mutually overlapping DTR sequences, including mini-satellites and micro-satellites in the centromere regions and G-quadruplexes (G4s) in the telomeres. Such secondary structures formed in DTRs, if not properly resolved, may block DNA replication fork movement and lead to genome instability. However, cells have developed mechanisms to resolve these barriers for efficient and faithful DNA replication. The most well-known way to resolve G4 and other secondary structures is unwinding the structure through DNA helicases. During the last funding period, we elucidated that the nuclease/helicase DNA2 facilitates DNA replication at DTR sequences. In our preliminary studies, we showed that the DNA mismatch repair protein MSH2, a component of the MutSα complex, binds to both G4s and DNA2 and strongly stimulates DNA2 to cleave G4 structures. Radiation, elimination of DNA2 or MSH2, or lack of histone H1c ubiquitination cause G4 accumulation. Therefore, we hypothesize that: 1) DNA2 in complex with MutSα excises and repairs G4 structures to facilitate DNA replication through DTRs; 2) ubiquitinated H1c recruits the DNA2/MutSα complex onto G4-bearing DNA ends at double- strand breaks (DSBs) for homology-directed DNA repair (HDR) of DSBs; and 3) gene mutations that impair G4 resolution processes sensitize individuals to chemicals that induce or stabilize G4 structures, leading to genome rearrangements and cancer initiation. We propose to define the important molecular aspects of the G4 excision pathways during DNA replication or DSB repair. Because G4s are implicated as chromosome structural elements and epigenetic motifs that regulate gene expression, G4 excision must be tightly controlled. It is important to elucidate how the DNA2/MutSα is signaled to be recruited to the G4 structure for excision repair. We will define how H1c ubiquitination mediated by ubiquitin E3 ligases ITCH or RNF8 induces DNA2/MutSα to cleave G4 structures during DNA replication and DSB repair. Moreover, a large number of environmentally contaminating compounds (ECCs) can specifically bind to G4 structures and alter the dynamics of G4 resolution. We expect that once genetic mutations impair a G4 resolution pathway, a G4 stabilizer can act synergistically to cause DNA replication stresses, DSBs, and genome rearrangements. Therefore, we will determine if combined genetic and environmental factors that inhibit proper G4 resolution/repair show synergy in promoting genome instability and cancer initiation. Our comprehensive analyses will define the pathway for G4 excision in S phase and during DNA DSB repair via HDR, and will provide evidence that G4-stabilizing ECCs lower the cancer threshold, particularly for individuals with defects in DNA2-mediated G4 and other secondary structure resolution pathways.