Pre-steady state kinetic studies show that an abasic site is a cognate lesion for the yeast Rev1 protein

Rev1 is a eukaryotic DNA polymerase that rescues replication forks stalled at sites of DNA damage by inserting nucleotides opposite the damaged template bases. Yeast genetic studies suggest that Rev1 plays an important role in rescuing replication forks stalled at one of the most common forms of DNA damage, an abasic site; however, steady state kinetic studies suggest that an abasic site acts as a significant block to nucleotide incorporation by Rev1. Here we examined the pre-steady state kinetics of nucleotide incorporation by yeast Rev1 with damaged and non-damaged DNA substrates. We found that yeast Rev1 is capable of rapid nucleotide incorporation, but only a small fraction of the protein molecules possessed this robust activity. We characterized the nucleotide incorporation by the catalytically robust fraction of yeast Rev1 and found that it efficiently incorporated dCTP opposite a template abasic site under pre-steady state conditions. We conclude from these studies that the abasic site is a cognate lesion for Rev1.

► The mechanisms of nucleotide incorporation by yeast and human Rev1 are similar.
► Only a small fraction of Rev1 molecules are capable of rapid nucleotide incorporation.
► Rev1s specificity comes mainly from the rate of the nucleotide-incorporation step.
► Nucleotide incorporation by Rev1 opposite abasic sites occurs very rapidly.
► Kinetics and in vivo evidence show that abasic sites are cognate lesions for Rev1.