Base excision repair (BER) is a major cellular pathway for DNA damage repair. During BER, DNA polymerase β (Polβ) is hypothesized to first perform gap-filling DNA synthesis by its polymerase activity and then cleave a 5′-deoxyribose-5-phosphate (dRP) moiety via its dRP lyase activity. In our recently accpeted PNAS paper, we demonstrated that gap-filling DNA synthesis by the polymerase activity likely occurred after Schiff base formation but before β-elimination, the two chemical reactions catalyzed by the dRP lyase activity, through gel-electrophoresis and kinetic analysis of partial BER reconstitution. The Schiff base formation and β-elimination intermediates were trapped by sodium borohydride reduction and identified by mass spectrometry and X-ray crystallography. Pre-steady-state kinetic analysis revealed that crosslinked Polβ (<i>i.e. </i>reduced Schiff base) exhibited a 17-fold higher polymerase efficiency than uncrosslinked Polβ. Conventional and time-resolved X-ray crystallography of crosslinked Polβ visualized important intermediates for its dRP lyase and polymerase activities, leading to a modified chemical mechanism for the dRP lyase activity. The observed interlocking enzymatic activities of Polβ allow us to propose an altered mechanism for the BER pathway, at least under the conditions employed. Plausibly, the temporally coordinated activities at the two Polβ active sites may well be the reason why Polβ has both active sites embedded in a single polypeptide chain. This proposed pathway suggests a new facet of BER and DNA repair, and may enable new chemical strategies for therapeutic intervention, as Polβ dysfunction is a key element common to several disorders.

Interlocking activities of DNA polymerase beta in the revised DNA base excision repair pathway

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