The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

Structures of LIG1 that engage with mutagenic mismatches inserted by polβ in base excision repair

“…1d ). All double-stranded DNA substrates were prepared by annealing upstream, downstream, and template primers 17 – 28 .…”

“…Human his-tag full-length (1-918) wild-type LIG1 and C-terminal (Δ261) E346A/E592A (EE/AA) LIG1 mutant were overexpressed in Rosetta (DE3) pLysS E. coli cells (Millipore Sigma) and grown in Terrific Broth (TB) media with kanamycin (50 μg ml −1 ) and chloramphenicol (34 μg ml −1 ) at 37 °C 17 – 28 . Once the OD was reached 1.0, the cells were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) and the overexpression was continued overnight at 28 °C.…”

“…Human GST-tag full-length polβ was overexpressed in One Shot BL21(DE3) E. coli cells (Invitrogen) in LB media at 37 °C for 8 h, induced with 0.5 mM IPTG, and the overexpression was continued for overnight at 28 °C 17 – 28 . After cell lysis at 4 °C by sonication in the lysis buffer containing 1X PBS (pH 7.3), 200 mM NaCl, 1 mM DTT, cOmplete Protease Inhibitor Cocktail (Roche), the lysate was pelleted at 31,000 x g for 1 h and then clarified by centrifugation and filtration.…”

“…The ligation assays using the nick DNA substrates containing 3′-preinserted correct dA:T, mismatches dG:T and dA:C were performed to test the ligation efficiency of wild-type and EE/AA mutant of LIG1 (Supplementary Fig. 5a ) 17 – 28 . Briefly, the ligation assays were performed in the mixture containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, 100 µgml −1 BSA, 10% glycerol, and 500 nM DNA substrate in a final volume of 10 µl.…”

“…Furthermore, several of the cancer-associated polβ variants possess aberrant repair functions, such as reduced fidelity stemming from impaired discrimination against incorrect nucleotide incorporation, and expression of these variants in cells induces cellular transformation and genomic instability 15 , 16 . In our studies, we have found that polβ mismatch or oxidized nucleotide 7,8-dihydro-8′-oxo-dGTP (8-oxodGTP) insertion products can generate a problematic nick repair intermediate for the subsequent ligation step in the BER pathway 6 , 10 , 17 – 28 . These findings contribute to understanding the important molecular determinants that ensure accurate BER pathway coordination or result in the impaired handoff from polβ to DNA ligase at the downstream steps.…”