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(POL)发挥着 在防止染色体不稳定和防止肿瘤发生方面的关键作用。与复制型波尔不同， TLS POL具有较少的限制活性STIES，它们缺乏校对3‘→5’核酸外切酶活性。 因此，纯化的TLS POL以极低的保真度合成与DNA损伤相反的DNA。尽管 这一点，TLS在正常人类细胞(不是来自癌症)中以一种主要的无差错方式工作， 这个项目的总体目标是确定高保真的细胞过程和机制 由本质上高度容易出错的Y系列POL强加于TLS。利用基因，细胞， 生化和结构方法，我们将解决以下问题：(1)Y家族POL 与多蛋白质系中的其他蛋白质因子相关，这些蛋白质是否具有升高活性 TLS Pol的保真度？(2)无错误的整个Y家族Pol组合的蛋白质组成是什么 人类细胞中的TLS？(3)TLS的保真度是如何受多蛋白系组成的调节的？ (4)多蛋白成分的作用机制的分子基础是什么？ 乐团将高保真度强加给Y家族波尔？为了追问这些问题，我们确定了一些 在TLS中起作用的蛋白质因子，与Y-家族POL特别结合；包括在这些蛋白质中 WRN具有dna解旋酶和3‘→5’外切酶活性，wrnip1具有dna。 依赖于ATPase活性。这些活动如何有助于Y家族Pol的TLS的保真度 不同类型的DNA损伤将在包括全基因组在内的广泛突变研究中进行分析 测序。使用将TurboID融合到POLη的邻近标记，我们将确定是否存在 与Y-家族POL一起在TLS中发挥作用的其他蛋白质以及这些蛋白质中的活性 这些POL会影响TLS的保真度。用纯化的POLη或POL多蛋白集合进行的生化研究 POLι，我们将确定WRN 3‘→5’外切酶、WRN和WRNIP1ATPase以及任何其他新的 根据不同类型的DNA损伤，这些POL确定了TLS的高保真活性。从低温- EM研究纯化的POLη或POLι多蛋白集合，我们将从机械上确定 多蛋白质组分调节这些Y家族POL相对于DNA损伤的保真度。 总而言之，这些研究将确定多蛋白Y家族TLS复制酶的成分 在人体细胞中进行高保真TLS。它们将揭示不同组件 限制Y-家族POL的活性部位以抑制核苷酸(NT)的错配以及WRN的3‘→5’是如何 核酸外切酶的活性与TLS Pol相协调，以去除错误插入的NT。这些研究将是 范式的转变，将为研究TLS Pol的忠诚度的机制细节开辟新的前景 将推动进一步阐述TLS POLS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.