Alkylation damage at the O6‐ and O4‐atoms of 2′‐deoxyguanosine (dG) and thymidine (T), respectively, can be removed by O6‐alkylguanine‐DNA alkyltransferases (AGTs). Previous studies have shown that human AGT (hAGT) repairs small adducts poorly at the O4‐atom of T, in comparison to the E. coli variants (OGT and Ada‐C). The C5 methyl group of the thymine nucleobase is suspected to contribute to hAGT repair proficiency possibly due to steric effects in the protein active site. In the present study, repair of oligonucleotides containing a 5‐fluoro‐O4‐methyl‐2′‐deoxyuridine (dFU‐Me) insert by hAGT, E. coli AGT variants (OGT and Ada‐C) and a chimeric hAGT/OGT protein was evaluated. All AGT variants, particularly hAGT and the hAGT/OGT chimera, demonstrated improved proficiency at removing the O4‐methyl group from substrates containing dFU‐Me, relative to the thymidine and 2′‐deoxyuridine counterparts.

Oligonucleotides containing various adducts, including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups, at the O atom of 5-fluoro-O -alkyl-2'-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5-fluoro-2'-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O -Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5-fluoro-O -benzyl-2'-deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O -ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogues of the O -alkylated nucleobases revealed that the C5-fluorine modification had an influence on reducing the electron density of the O -C bond, relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on the repair of larger O -alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.

The β-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the β-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have –almost exclusively– been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the β-clamp has different roles depending on the organism. In Bacillus subtilis , the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the β-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective β-clamps. The structures reveal different binding modes consistent with the binding to the β-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.

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