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

蛋白质磷酸化是真核细胞中最重要的翻译后修饰形式之一，有效的DNA修复机制对所有形式的生命都是必不可少的。我的研究计划涵盖了生物学的这两个重要领域，即蛋白激酶的作用和DNA修复的机制。 我的实验室的主要重点是了解丝氨酸/苏氨酸蛋白激酶，DNA依赖的蛋白激酶催化亚单位(DNA-PKcs/基因名称PRKDC)的功能。脊椎动物细胞需要DNA-PKcs来修复一种称为DNA双链断裂(DSB)的剧毒形式的DNA损伤。它通过非同源末端连接(NHEJ)的过程来实现这一点，NHEJ是脊椎动物DSB修复的主要途径，在NHEJ过程中，它与Ku70/80异二聚体和双链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途径的其他成分相互作用方面拥有丰富的经验。最近，一种名为LINP1的长非编码RNA被证明对NHEJ具有调节作用。在本应用(项目1)中，我们将确定双链RNA如何与Ku70/80和NHEJ途径的其他组件相互作用，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从人类细胞中删除了DNA-PKcs或相关的蛋白激酶共济失调毛细血管扩张突变(ATM)。在目前正在发表的工作中，我们已经表明DNA-PKcs的缺失会导致ATM蛋白水平的显著降低，并且缺乏DNA-PKcs的细胞具有戏剧性的有丝分裂表型。此外，我们还发现DNA-PKcs是细胞周期G2期细胞周期蛋白B1、芳香素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期早期调控细胞周期调控基因表达中的新作用。 在本申请的项目2中，我们将确定DNA-PKcs如何调控ATM的表达，特别是询问ATM的调控是通过转录、翻译还是翻译后修饰来调节的。 在项目3中，我们将确定DNA-PKcs通过B-Myb、FOXM1、MuvB转录因子的磷酸化和/或激活来调节细胞周期调节的有丝分裂基因的表达的机制，如细胞周期调节基因，如细胞周期蛋白B1、芳香素和Securin在细胞周期的G2期。 这项研究计划将确定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。有了独特的试剂和令人兴奋的初步数据，我们将处于独特的地位，可以揭示DNA-PKcs的新修复和非修复功能，并在未来几年引领该领域对这种耐人寻味的蛋白激酶的生物学的了解。

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.