Repair and non-repair functions of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 的修复和非修复功能

• 批准号: RGPIN-2019-04829
• 负责人: LeesMiller, Susan
• 金额: $ 3.64万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737888
• 关键词: Repair; non; repair; functions; DNA

Protein phosphorylation is one of the most important forms of post-translational modification in eukaryotic cells and efficient DNA repair mechanisms are essential to all forms of life. My research program encompasses both of these important areas of biology, the role of protein kinases and mechanisms of DNA repair. The main focus of my laboratory is understanding the function of the serine/threonine protein kinase, DNA-dependent protein kinase catalytic subunit (DNA-PKcs/gene name PRKDC). DNA-PKcs is required in vertebrate cells for the repair of a highly toxic form of DNA damage called a DNA double strand break (DSB). It does this through the process of non-homologous end joining (NHEJ), the main pathway for repair of DSBs in vertebrates,  where it interacts with the Ku70/80 heterodimer and double-stranded DNA to form the active protein kinase, DNA-PK. My lab has extensive experience in studying how DNA-PKcs functions in NHEJ and how it interacts with double stranded DNA, Ku and other components of the NHEJ pathway. Recently, a long non-coding RNA called LINP1 has been shown to regulate NHEJ. In this application (Project 1), we will determine how double stranded RNA interacts with Ku70/80 and other components of the NHEJ pathway and whether double stranded RNA regulates DNA-PK activity. To better understand the roles of DNA-PK in cells, we used CRISPR-Cas9 to delete DNA-PKcs or the related protein kinase Ataxia Telangiectasia Mutated (ATM) from human cells. In work that is currently being written up for publication, we have shown that deletion of DNA-PKcs causes a significant reduction in the level of ATM protein and that cells lacking DNA-PKcs have a dramatic mitotic phenotype. Moreover, we have shown that DNA-PKcs is required for the expression of cyclin B1, anillin and other mitotic proteins in G2 phase of the cell cycle.  Thus, we have uncovered a novel role for DNA-PKcs in controlling the expression of cell cycle regulatory genes in early G2 phase. In project 2 of this application, we will determine how DNA-PKcs regulates the expression of ATM, specifically asking whether regulation of ATM is mediated by transcription, translation or post translational modification. In project 3 we will determine the mechanism by which DNA-PKcs regulates the expression of cell cycle regulated mitotic genes such as cyclin B1, anillin and securin in G2 phase of the cell cycle through phosphorylation and/or activation of B-Myb, FOXM1, MuvB transcription factors.  This program of research will determine how RNA regulates NHEJ and elucidate novel functions of DNA-PKcs in transcription and regulation of cell cycle genes. With the unique reagents and the exciting preliminary data that we have generated, we are in a unique position to uncover novel repair and non-repair functions of DNA-PKcs and to lead the field in understanding the biology of this intriguing protein kinase for years to come.

蛋白质磷酸化是真核细胞翻译后修饰的最重要形式之一，有效的DNA修复机制对所有形式的生命都是必不可少的。我的研究项目包括这两个重要的生物学领域，蛋白激酶的作用和DNA修复机制。我实验室的主要重点是了解丝氨酸/苏氨酸蛋白激酶，dna依赖性蛋白激酶催化亚基（DNA-PKcs/基因名称PRKDC）的功能。在脊椎动物细胞中，DNA- pkcs是修复一种称为DNA双链断裂（DSB）的高毒性DNA损伤所必需的。它通过非同源末端连接（NHEJ）过程来完成这一过程，这是脊椎动物修复dsb的主要途径，它与Ku70/80异源二聚体和双链DNA相互作用，形成活性蛋白激酶DNA- pk。我的实验室在研究DNA- pkcs如何在NHEJ中发挥作用以及它如何与双链DNA、Ku和NHEJ途径的其他组分相互作用方面具有丰富的经验。最近，一种名为LINP1的长链非编码RNA被证明可以调节NHEJ。在本应用程序（项目1）中，我们将确定双链RNA如何与Ku70/80和NHEJ通路的其他组分相互作用，以及双链RNA是否调节DNA-PK活性。为了更好地了解DNA-PK在细胞中的作用，我们使用CRISPR-Cas9从人类细胞中删除DNA-PKcs或相关蛋白激酶Ataxia telangiecasia Mutated （ATM）。在即将发表的研究中，我们发现DNA-PKcs的缺失会导致ATM蛋白水平的显著降低，并且缺乏DNA-PKcs的细胞具有显著的有丝分裂表型。此外，我们已经证明DNA-PKcs是细胞周期G2期cyclin B1、anillin和其他有丝分裂蛋白表达所必需的。因此，我们发现了DNA-PKcs在G2期早期控制细胞周期调控基因表达中的新作用。在本项目的项目2中，我们将确定DNA-PKcs是如何调控ATM的表达的，特别是探究ATM的调控是通过转录、翻译还是翻译后修饰介导的。在项目3中，我们将通过磷酸化和/或激活B-Myb、FOXM1、MuvB转录因子，确定DNA-PKcs在细胞周期G2期调控细胞周期调控的有丝分裂基因（如cyclin B1、anillin和securin）表达的机制。本研究项目将确定RNA如何调控NHEJ，并阐明DNA-PKcs在细胞周期基因转录和调控中的新功能。凭借独特的试剂和令人兴奋的初步数据，我们处于一个独特的位置，可以发现DNA-PKcs的新的修复和非修复功能，并在未来几年领导该领域了解这种有趣的蛋白激酶的生物学。