Hydrogen-Bonding Interactions at the DNA Terminus Promote Extension from Methylguanine Lesions by Human Extender DNA Polymerase ζ.

Abstract

Chemically induced DNA lesions can become DNA replication substrates that are bypassed by low-fidelity DNA polymerases. Following nucleotide misinsertion opposite a DNA lesion, the extension step can contribute to preserving such errors and lead to genomic instability and cancer. DNA polymerase ζ, a B-family polymerase, is proficient as an extender polymerase that catalyzes elongation; however, the chemical factors that impact its DNA replication are not understood. This study addresses the question of how DNA polymerase ζ achieves extension by examining the ability of recombinant human DNA polymerase ζ to extend from a series of methylated guanine lesions. The influence of H-bonding was examined by placing structurally altered nucleoside analogues and canonical bases opposite G, O6-MeG, N1-MeG, and N2-MeG. We determined that terminal base pairs with the highest proclivity for H-bonding were most efficiently extended in both primer extension assays and steady-state kinetic analysis. In contrast, when no H-bonding was possible at the DNA terminus, the least efficient steady-state kinetics were observed. To evaluate H-bonding protein minor groove interactions that may underlie this phenomenon, we performed computational modeling with Escherichia coli DNA polymerase II, a homologue for DNA polymerase ζ. The modeling data together with the primer extension assays demonstrate the importance of having a carbonyl group on the primer strand that can interact with a lysine residue found to be conserved in many B-family polymerases, including human Pol ζ. These data provide a model whereby interbase H-bonding interactions at the DNA terminus promote lesion bypass and extension by human DNA polymerase ζ.