Structural and Molecular Basis of Polq Functions in DNA repair

DNA polymerase theta (Polθ, encoded by POLQ) is a unique multidomain enzyme essential for alternative double-strand break (DSB) repair and cellular resistance to genotoxic stress. It is the only known human enzyme containing both a helicase (Polθ-h) and a polymerase (Polθ-p) domain, which together mediate error- prone microhomology-mediated end joining (MMEJ), translesion synthesis (TLS), and single-strand gap repair. Polθ is highly expressed in homologous recombination-deficient (HRD) tumors and synthetic lethal with BRCA1/2 loss, making it a promising therapeutic target. However, the structural mechanisms underlying its DNA repair activities and their essentiality in HRD cancer cells remain unclear. This project aims to elucidate the structural basis of Polθ’s TLS and MMEJ functions and define their biological importance in HRD contexts. Three specific aims are designed to achieve our goals. Aim 1 will determine how Polθ-p performs TLS across UV-induced lesions using high-resolution X-ray crystallography and cryoEM. Aim 2 will resolve how Polθ promotes MMEJ by solving cryoEM structures of Polθ complexes with 3′ ssDNA overhangs and characterizing domain cooperativity. Aim 3 will identify which Polθ activities are essential for BRCA2-deficient cell survival using structure-function mutants and cellular assays. By integrating structural biology, biochemistry, and cell biology, this study will define how Polθ mediates error-prone DNA repair and supports HRD cell survival. The findings will provide a mechanistic framework for exploiting Polθ as a cancer therapeutic target and may inform the rational design of next-generation Polθ inhibitors. Project Number: 1R01GM164504-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: XIAOJIANG CHEN | Institution: UNIVERSITY OF SOUTHERN CALIFORNIA, Los Angeles, CA | Award Amount: $506,018 | Activity Code: R01 | Study Section: Macromolecular Structure and Function A Study Section[MSFA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11343684