Dynamic functions of DNA2 counteract DNA replication stresses and tumorigenesis

摘要 癌细胞的产生和发展是由于基因和表观遗传改变的积累导致的 癌症表型。这些变化的一个潜在原因是dna序列“难以复制”。 (DTR)，并作为复制应激源的内源。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(G4S)。这种在DRR中形成的二级结构，如果没有得到适当的解决， 可能会阻碍DNA复制分叉运动，导致基因组不稳定。然而，细胞已经发展起来 解决这些障碍的机制，以实现高效和忠实的DNA复制。最广为人知的 分解G4和其他二级结构是通过DNA解旋酶来解开结构。在过去的几年里 在融资期间，我们阐明了核酸酶/解旋酶DNA2促进DTR序列上的DNA复制。在……里面 我们的初步研究表明，错配修复蛋白msh2是MutSα的一个组成部分 复合体，与G4S和DNA2结合，并强烈刺激DNA2裂解G4结构。辐射， DNA2或MSH2的消除或缺乏组蛋白H1c泛素化会导致G4积聚。因此，我们 假设：1)与MutSα形成的复合体中的DNA切除并修复G4结构以促进DNA复制 2)泛素化的H1c将DNA 2/MutsDNA复合体招募到含G4的α末端。 链断裂(DSB)，用于DSB的同源定向DNA修复(HDR)；3)损害G4的基因突变 分解过程使个体对诱导或稳定G4结构的化学物质敏感，从而导致基因组 重排和癌症的发生。我们建议定义G4切除的重要分子方面 DNA复制或DSB修复过程中的通路。因为G4S与染色体结构元素有关 以及调控基因表达的表观遗传基序，必须严格控制G4切除。重要的是要 阐明DNA 2/MutSα是如何被征募到G4结构中进行切除修复的。我们将定义 泛素E3连接酶介导的H1c泛素化如何诱导DNA_2/MutSα裂解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(ECC)可以与G4结构特异性结合，改变G4的拆分动力学。我们预计 一旦基因突变损害了G4分解途径，G4稳定剂就可以协同作用，导致DNA 复制应激、双链断裂和基因组重排。因此，我们将确定联合遗传和 抑制G4正确分辨/修复的环境因素在促进基因组不稳定和 癌症的开端。我们的综合分析将确定G4切除的途径在S阶段和 通过HDR修复DNA DSB，并将提供证据表明G4稳定的ECC降低癌症阈值， 尤其是在DNA2介导的G4和其他二级结构解析途径中存在缺陷的个体。

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.