Interlocking activities of DNA polymerase β in the base excision repair pathway

Base excision repair (BER) is a major cellular pathway for DNA damage repair. During BER, DNA polymerase beta (Pol beta) 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. Through gel electrophoresis and kinetic analysis of partial BER reconstitution, we demonstrated that gap-filling DNA synthesis by the polymerase activity likely occurred after Schiff base formation but before beta-elimination, the two chemical reactions catalyzed by the dRP lyase activity. The Schiff base formation and beta-elimination intermediates were trapped by sodium borohydride reduction and identified by mass spectrometry and X-ray crystallography. Presteady-state kinetic analysis revealed that cross-linked Pol beta (i.e., reduced Schiff base) exhibited a 17-fold higher polymerase efficiency than uncrosslinked Pol beta. Conventional and time-resolved X-ray crystallography of cross-linked Pol beta 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 Pol beta 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 Pol beta active sites may well be the reason why Pol beta has both active sites embedded in a single polypeptide chain. This proposed pathway suggests a corrected facet of BER and DNA repair, and may enable alternative chemical strategies for therapeutic intervention, as Pol beta dysfunction is a key element common to several disorders.

Automated summary

Base excision repair (BER) is a crucial cellular pathway for DNA damage repair, and DNA polymerase beta (Pol beta) plays a key role in this process. This study reveals the sequential order of two enzymatic activities of Pol beta during BER and identifies the intermediate molecules involved. The coordinated activities of Pol beta's active sites are found to be critical for the BER pathway, suggesting potential alternative strategies for therapeutic intervention.