*4.4. DNA Synthesis by Pol*β

The activity of Polβ in DNA synthesis was assayed in reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 6 mM MgCl2, 50 nM 5'-32P-labelled gap-DNA substrate (32 base-pair oligonucleotide with a one-nucleotide gap prepared as described in Supplementary Materials), and 50 nM Polβ. Reaction mixtures were preassembled on ice; when indicated, they were supplemented with APE1, APE1N∆61, and XRCC1 (at concentrations specified in the figure legends). The reaction was initiated by adding a mixture of dATP (2'-deoxyadenosine 5'-triphosphate), dGTP (2'-deoxyguanosine 5'-triphosphate), dTTP (2'-deoxythymidine 5'-triphosphate) and dCTP (2'-deoxycytidine 5'-triphosphate) to a final concentration of 10 µM for each nucleotide. Reaction mixtures were incubated at 37 ◦C for 30 min, and terminated by the addition of denaturing PAGE sample buffer and heating for 2 min at 90 ◦C. The reaction products were separated by electrophoresis in 20% denaturing polyacrylamide gels. The gels were imaged on a Typhoon FLA 9500, and the amounts of DNA substrate and products were quantified using Quantity One Basic software.
