Human DNA polymerase κ forms nonproductive complexes with matched primer termini but not with mismatched primer termini

Abstract

Human DNA polymerase kappa (pol κ) is a member of the Y family of DNA polymerases that function in translesion synthesis. It synthesizes DNA with moderate fidelity and does not efficiently incorporate nucleotides opposite DNA lesions. Pol κ has the unusual ability to efficiently extend from mismatched primer termini, and it extends readily from nucleotides inserted by other DNA polymerases opposite a variety of DNA lesions. All of this has suggested that pol κ functions during the extension step of translesion synthesis. Here, we have carried out pre-steady-state kinetic studies of pol κ using DNA with matched and mismatched primer termini. Interestingly, we find that mismatches present only a modest kinetic barrier to nucleotide incorporation by pol κ. Moreover, and quite surprisingly, active-site titrations revealed that the concentration of active pol κ is very low with matched DNA, and from DNA trapping experiments we determined that this was due to the formation of nonproductive protein·DNA complexes. In marked contrast, we found that the concentration of active pol κ was six-fold greater with mismatched DNA than with matched DNA. Thus, pol κ forms nonproductive complexes with matched but not with mismatched DNA. From these observations, we conclude that pol κ has evolved to specifically function on DNA substrates with aberrant primer-terminal base pairs, such as the ones it would encounter during the extension step of translesion synthesis.

• DNA damage
• DNA repair
• DNA replication
• kinetics
• mutagenesis