Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase lambda.

Abstract:

DNA polymerase lambda (Pol lambda) is a novel X-family DNA polymerase ...
DNA polymerase lambda (Pol lambda) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase beta. Pre-steady-state kinetic studies have shown that the Pol lambda-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase beta-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol lambda's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol lambda to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol lambda binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate>>base>ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K(d). The fidelity of Pol lambda was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.

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