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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 et al ., 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 in vitro assay enables us to measure the products for correct vs. oxidized nucleotide insertion, DNA ligation, and ligation failure, i.e ., 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 in vitro .

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