(A) Agarose gel electrophoresis of the native form of GC, mGC, GC+MMS, and mGC+MMS. (B) Restriction map of pAS201; the two single cutter enzymes BmtI and BaeGI yield a short S (589 bp) and a long L (1525 bp) fragment, respectively. Fragment S contains the single O6mG adduct. (C) Plasmids GC, mGC, GC+MMS, and mGC+MMS were incubated in nucleoplasmic extracts (NPE) in the presence of α32P-dATP and extracted after 2 hr. The purified plasmids were digested with restriction enzymes BmtI and BaeGI. Digested plasmids were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining and by 32P imaging. (D) The specific activity (SA) of a given fragment (S or L) was quantified as the amount of 32P incorporated divided by the amount of DNA deduced from the ethidium bromide fluorescence image (expressed in arbitrary units [AU]). In control plasmid GC, a similar SA value was observed for both fragments as expected for residual background incorporation; similarly, in methyl-methane sulfonate (MMS)-treated plasmids (GC+MMS), the SA value was similar for S and L fragments. The slightly increased SA value in GC+MMS compared to GC is compatible with repair synthesis by base excision repair (BER) at randomly located MMS-induced lesions. For plasmids containing a single O6mG:C lesion (mGC and mGC+MMS), there is a robust increase in SA of the short compared to the long fragments. Incorporation, above background, due to O6mG, in the absence of MMS, amounts to 0.065 and 0.495 AU for long and short fragments, respectively (signal above the dotted line in S3D). Similarly, in the context of MMS lesions, incorporation, above background, due to O6mG, amounts to 0.115 and 1.17 AU for long and short fragments, respectively (signal above the dotted line in S3D). Taken together, these results clearly show that O6mGC-mediated repair specifically takes place in the S fragment, with only modest spill-over into the L fragment (10–15%). (E) Plasmids with a single O6mG:C lesion site (mGC) and control plasmids (GC) were incubated in NPE depleted with anti-Mlh1 antibodies and mock-treated NPE. Incorporation was measured into whole plasmids. The increase in incorporation into mGC compared to GC observed upon incubation in NPE fully disappeared when Mlh1 was depleted. This experiment shows that the increase in incorporation observed in mGC compared to incorporation in control GC plasmid can specifically be attributed to mismatch repair activity at the single O6mG:C lesion.