ABSTRACT
Base Excision Repair (BER) is carried out by a series of DNA repair proteins that function in a step-by-step process to identify, remove, and replace DNA damage. As DNA damage is processed during BER, the DNA transitions through various intermediate states, called BER intermediates, which if left exposed can develop into double-strand DNA breaks and trigger programmed cell death signaling. Previous studies have proposed that in order to minimize exposure of the BER intermediates, each protein may remain bound to its product prior to the next protein binding. Thus, a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme may form between each step of the BER pathway. The transfer of BER intermediates between enzymes, known as BER coordination, has yet to be directly visualized and the mechanistic details of the process remain unclear. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β). When preformed complexes comprised of APE1 and the incised AP-site product were subsequently bound by Pol β, the Pol β enzyme dissociated shortly after binding in a majority of the observations. In the events where Pol β binding was followed by APE1 dissociation (i.e., DNA hand-off), Pol β had remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that, in the absence of other BER factors, transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration that Pol β remains bound near the APE1-5’ nick complex. These findings provide insight into how APE1 and Pol β coordinate the transfer of DNA within the BER pathway.
Competing Interest Statement
The authors have declared no competing interest.
