XRCC1 Enables the Efficient Local Search for DNA Damage by DNA Polymerase Beta
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 ꞵ) 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 ꞵ to coordinate BER factors at DNA damage. XRCC1 enhances BER efficiency, though the mechanism by which this occurs is unclear. Pol β 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 ꞵ recruitment, particularly XRCC1, remains unclear. Here, we used correlative optical tweezers-fluorescence microscopy to visualize DNA search and damage recognition by pol ꞵ and XRCC1. We characterize each factor individually, examine their behavior as the pol ꞵ-XRCC1 complex, and assess their interplay with apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme upstream of pol ꞵ in BER. We find that pol ꞵ locates damage through 3D-diffusion, whereas XRCC1 exhibits both 3D- and 1D-diffusion. In combination, XRCC1 dramatically shifts pol β search towards 1D-diffusion, enabling interrogation of non-damaged DNA using both search mechanisms. When both APE1 and pol ꞵ are present, the pol ꞵ-1nt gap complex is highly stable, with APE1 largely unable to disrupt the damage-bound pol ꞵ. Together, these findings demonstrate that XRCC1 reshapes pol β 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. Significance Statement DNA repair proteins must locate rare sites of damage hidden within millions of undamaged bases. Using single-molecule imaging with optical tweezers, we directly visualized how DNA polymerase ꞵ and its scaffold partner XRCC1 search for and engage DNA damage. Alone, pol β finds damage exclusively through 3D collisions, whereas XRCC1 scans along DNA by 1D hopping. When the two proteins form a complex, XRCC1 confers its scanning ability on pol β, expanding the search strategies available for damage detection. These findings reveal a mechanism by which scaffold proteins remodel the damage search process of their partners, providing insight into how base excision repair is coordinated to maintain genome stability.