Mechanisms for the high fidelity of translesion synthesis by Y-family DNA polymerases in human cells

摘要 通过促进DNA损伤的复制，跨损伤合成（TLS）DNA聚合酶（Pos）发挥了重要作用。 在防止染色体不稳定性和防止肿瘤发生中起关键作用。与复制型波尔不同， TLS Pol具有较少限制的活性位点，并且它们缺乏校正3 '→ 5'外切核酸酶活性。 因此，纯化的TLS Pol以极低的保真度合成与DNA损伤相对的DNA。尽管 TLS在正常人类细胞（不是来自癌症）中以主要无错误的方式运行， 本项目的总体目标是确定高保真度的细胞过程和机制， 由本质上高度易错的Y族Pol施加在TLS上。利用基因，细胞， 生物化学和结构的方法，我们将解决以下问题：（1）做Y-家族的Pos 与多蛋白质集合中的其他蛋白质因子相关联，这些蛋白质是否具有提高 TLS Pol的保真度？(2)什么是蛋白质组成的整个Y-家庭Pol合奏为无误 人类细胞中的TLS？(3)TLS的保真度是如何被多蛋白系综的组分所调节的？ (4)作用机制的分子基础是什么？ 集成使Y系列柱体具有高保真度？为了解决这些问题，我们确定了一些 在TLS中特异性与Y家族Pol结合起作用的蛋白质因子;包括在这些蛋白质中 WRN具有DNA解旋酶和3 '→ 5'核酸外切酶活性，WRNIP1具有DNA解旋酶和3 '→ 5'核酸外切酶活性。 依赖ATP酶活性。这些活动如何通过Y族Pol对TLS的保真度做出贡献， 不同类型的DNA损伤将在广泛的突变研究中进行分析，包括全基因组研究， 测序使用邻近标记，其中TurboID与Pol η融合，我们将确定是否存在 在TLS中与Y家族Pol结合起作用的其他蛋白质，以及这些蛋白质中的活性是否 影响TLS的保真度。在用纯化的Pol η或 Pol 1，我们将确定WRN 3 '→ 5'外切核酸酶，WRN和WRNIP1 ATP酶，以及任何其他新的 通过这些Pol在TLS的高保真度中鉴定出与不同类型的DNA损伤相对的活性。从低温- 通过使用Pol η或Pol ι的纯化多蛋白系综的EM研究，我们将从机理上确定 多蛋白系综的组分调节这些Y家族Pol相对于DNA损伤的保真度。 累积起来，这些研究将鉴定多蛋白Y家族TLS复制酶的组分， 在人体细胞中进行高保真TLS。它们将揭示各种成分 限制Y家族Pos的活性位点以抑制核苷酸（nt）的错误掺入以及WRN的3 '→ 5' 外切核酸酶活性与TLS Pol协调以去除错误插入的nt。这些研究报告将 范式转变，并将开辟新的前景的研究机制的细节TLS Pos的保真度，他们 将推动进一步阐述TLS Pol在基因组完整性中的作用。

ABSTRACT By promoting replication through DNA lesions, translesion synthesis (TLS) DNA polymerases (Pols) play a critical role in preventing chromosomal instability and protecting against tumorigenesis. Unlike replicative Pols, TLS Pols have less constrained active sties and they lack proofreading 3'→5' exonuclease activity. Consequently, purified TLS Pols synthesize DNA opposite DNA lesions with an extremely low fidelity. Despite this, TLS operates in a predominantly error-free manner in normal human cells (not derived from cancers), The overall objective in this project is to identify the cellular processes and mechanisms by which high fidelity is imposed upon TLS by the intrinsically highly error-prone Y-family Pols. Using a combination of genetic, cellular, biochemical, and structural approaches, we will address the following questions: (1) Do the Y-family Pols associate with other protein factors in a multiprotein ensemble and do these proteins have activities that elevate the fidelity of the TLS Pol? (2) What is the protein composition of the entire Y-family Pol ensemble for error-free TLS in human cells? (3) How is the fidelity of TLS modulated by the components of the multiprotein ensemble? (4) What are the molecular underpinnings of action mechanisms via which components of the multiprotein ensemble impose high fidelity on Y-family Pols? To pursue these questions, we have identified a number of protein factors that function in TLS specifically in conjunction with Y-family Pols; included among these proteins are WRN which possesses DNA helicase and 3'→5' exonuclease activities, and WRNIP1 which has a DNA dependent ATPase activity. How these activities contribute to the fidelity of TLS by Y-family Pols opposite different types of DNA lesions will be analyzed in extensive mutational studies that include genome wide sequencing. Using proximity labeling in which TurboID is fused to Polη, we will determine whether there are additional proteins that function in TLS in conjunction with Y-family Pols and whether activities in these proteins affect the fidelity of TLS by these Pols. In biochemical studies with the purified multiprotein ensemble of Polη or Polι, we will ascertain the roles of WRN 3'→5' exonuclease, WRN and WRNIP1 ATPase, and of any other newly identified activities in the high fidelity of TLS by these Pols opposite different types of DNA lesions. From cryo- EM studies with the purified multiprotein ensemble of Polη or Polι, we will determine mechanistically how the components of the multiprotein ensemble modulate the fidelity of these Y-family Pols opposite DNA lesions. Cumulatively, these studies will identify the components of the multiprotein Y-family TLS replicases which carry out high fidelity TLS in human cells. They will reveal the mechanisms by which the various components constrain Y-family Pols' active sites to restrain nucleotide (nt) misincorporation and how WRN's 3'→5' exonuclease activity is coordinated with the TLS Pol for the removal of misinserted nt. These studies will be paradigm shifting and will open new vistas of research into the mechanistic details of TLS Pols' fidelity and they will give impetus to further elaboration of the roles of TLS Pols in genome integrity.