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。

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.