Helicases: Getting Down to the Bottom of DNA with AfXPB

Helicases play a central role in genome maintenance by unwinding nucleic acids and facilitating replication, repair, and transcription. The archaeal XPB helicase family operates through ATP-driven translocation and strand separation. "Molecular wrench" activity has been observed in the Archaeoglobus fulgidus XPB helicase (AfXPB), characterized by rapid exponential DNA unwinding triggered by a 170° ATP-induced domain rotation. To investigate how DNA sequence integrity influences helicase–DNA binding affinity, we examined AfXPB interactions with a 17-nucleotide double-stranded DNA probe containing either a fully complementary sequence or a single mismatched base. Binding was quantified using helicase concentration titration experiments, from which dissociation constants were determined. Our results reveal that AfXPB exhibits a smaller dissociation constant when interacting with the mismatched DNA sequence compared to the well-matched control, indicating higher binding affinity. These findings suggest that even a single base mismatch can be recognized by AfXPB for better DNA repair. This work highlights the sensitivity of AfXPB to local sequence disruptions and provides insight into how mismatches may influence helicase activity in DNA repair pathways.