Pol?/XRCC1 heterodimerization dictates DNA damage recognition and basal Pol? protein levels without interfering with mouse viability or fertility

DNA Polymerase beta (Pol beta) performs two critical enzymatic steps during base excision repair (BER) - gap filling (nucleotidyl transferase activity) and gap tailoring (dRP lyase activity). X-ray repair cross complementing 1 (XRCC1) facilitates the recruitment of Pol beta to sites of DNA damage through an evolutionarily conserved Pol beta/ XRCC1 interaction interface, the V303 loop. While previous work describes the importance of the Pol beta/XRCC1 interaction for human Pol beta protein stability and recruitment to sites of DNA damage, the impact of disrupting the Pol beta/XRCC1 interface on animal viability, physiology, and fertility is unknown. Here, we characterized the effect of disrupting Pol beta/XRCC1 heterodimerization in mice and mouse cells by complimentary approaches. First, we demonstrate, via laser micro-irradiation, that mouse Pol beta amino acid residues L301 and V303 are critical to facilitating Pol beta recruitment to sites of DNA damage. Next, we solved the crystal structures of mouse wild type Pol beta and a mutant protein harboring alterations in residues L301 and V303 (L301R/V303R). Our structural analyses suggest that Pol beta amino acid residue V303 plays a role in maintaining an interaction with the oxidized form of XRCC1. Finally, we created CRISPR/Cas9-modified Polb mice with homozygous L301R/V303R mutations (Polb(L301R-V303R/L301R-V303R)) that are fertile yet exhibit 15% reduced body weight at 17 weeks of age, as compared to heterozygous mice. Fibroblasts derived from Polb(L301R-V303R/L301R)-V303R mice demonstrate that mutation of mouse Pol beta's XRCC1 interaction domain leads to an similar to 85% decrease in Pol beta protein levels. In all, these studies are consistent with a role for the oxidized form of XRCC1 in providing stability to the Pol beta protein through Pol beta/XRCC1 heterodimer formation.