Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase β during base excision repair.

Base excision repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol β substrate) were subsequently bound by Pol β, the Pol β enzyme dissociated shortly after binding in most of the observations. In the events where Pol β binding was followed by APE1 dissociation during substrate channeling, Pol β remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

碱基切除修复（BER）由一系列蛋白质执行，这些蛋白质按逐步的过程发挥作用，以识别、去除和替换DNA损伤。在BER过程中，DNA在被每种DNA修复酶处理时会经历各种中间状态。如果不进行修复，这些BER中间产物可转变为双链DNA断裂，并促进基因组不稳定。先前的研究提出，在BER途径的每一步都存在一种由BER中间产物、进入的酶和离开的酶组成的短暂复合物，以保护BER中间产物。酶之间BER中间产物的转移，即BER协同作用或底物通道化，仍然知之甚少。在此，我们利用单分子全内反射荧光显微镜来研究无嘌呤/无嘧啶核酸内切酶1（APE1）和DNA聚合酶β（Pol β）之间BER协同作用的机制。当APE1与切割后的无碱基位点产物（APE1产物和Pol β底物）预先形成的复合物随后被Pol β结合时，在大多数观察结果中，Pol β酶在结合后不久就解离。在底物通道化过程中，Pol β结合后接着APE1解离的情况下，Pol β会保持结合较长时间以允许APE1解离。我们的结果表明，在BER过程中BER中间产物从APE1到Pol β的转移取决于APE1的解离动力学以及三元复合物在切割后的无碱基位点上的持续时间。