Distinct mechanisms of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

UV-light-induced cyclobutane pyrimidine dinners (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Pol eta promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Pol eta, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Pol eta. The x-ray crystal structure of Dpo4 complexed with either the 3'-thymine (T) or the 5' T of a cis-syn TT dinner has indicated that, whereas the 3' T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5' T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5'T of the dimer with the incoming A has been proposed for Pol eta. Here we examine the mechanisms of CPD bypass by Dpo4 and Pol eta using nucleoticle analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Pol eta incorporate dATP opposite the 5' T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3' T of the dinner, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Pol eta using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.