A Thymine Dimer Stalls a High-Fidelity DNA Polymerase by Providing No Template Information in the Same Manner as an...

Cyclobutane pyrimidine dimers are photolesions that form when UV-B irradiation causes adjacent thymine or cytosine bases to covalently bond and can arrest DNA synthesis by replicative polymerases. To gain insight into the effects of cis-syn cyclobutane thymine dimers (T=T) on DNA replication in a model polymerase, we conducted solution and crystallographic studies of the Bacillus DNA polymerase I large fragment (BF) in complex with a T=T-containing template and an incoming dNTP. A thymine dimer lesion blocked nucleotide addition by BF in solution. The crystal structure of the BF-T=T-dATP complex indicated that the thymine dimer is too large to enter the template insertion site, preventing the lesion from providing any information to copy. We compare the T=T ternary complex with a BF ternary complex with an abasic site analogue and show that both lesions are noninstructional and induce nearly identical conformations in the polymerase and the substrate dATP. The incoming dATP inserts too far into the active site in the absence of a template base and distorts into a structure that is not conducive for catalysis with the primer 3'-hydroxyl. Together, our results show that thymine dimers and abasic sites can stall DNA synthesis by providing an absence of information through similar mechanisms.