Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair.

We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan ., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase l. This assay enables us to measure the products for correct vs. oxidized nucleotide insertion, DNA ligation, and ligation failure, ., abortive ligation products, as a function of reaction time. This protocol complements our previous publication and describes an efficient way to analyze activities of BER enzymes and the functional interaction between pol β and DNA ligase I .