XRCC1 Enables the Efficient Local Search for DNA Damage by DNA Polymerase Beta

Abstract

Oxidative DNA damage is a common threat to genomic integrity, arising from endogenous metabolic processes and environmental exposures. If unrepaired, such oxidative DNA damage promotes mutagenesis and genomic instability. Cells counter this through base excision repair (BER), a multi-step pathway requiring the coordinated action of several proteins. Central to BER, DNA polymerase beta (pol ) locates single-nucleotide (1-nt) gaps and inserts the correct nucleotide, while x-ray repair cross-complementing 1 (XRCC1) is a scaffold protein that forms a stable complex with pol to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol {beta} is proposed to be recruited to DNA damage by undamaged DNA scanning interactions, but this behavior has not yet been directly observed. Additionally, the influence of other BER proteins on pol recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol and XRCC1. We characterize each factor individually, examine their behavior as the pol -XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol in BER. We find that pol locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol {beta} search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol are present, the pol -1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol . Together, these findings demonstrate that XRCC1 reshapes pol {beta} search behavior to promote efficient local damage recognition, providing a mechanistic basis for how BER factors coordinate lesion detection and processing to maintain genomic stability.