Bypass of an Abasic Site via the A-Rule by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

Abstract

The abasic site is one the most common DNA lesions formed in cells; it induces a severe blockage of DNA replication and is highly mutagenic. We continue to use Gp90 exo-, the sole DNA polymerase from Pseudomonas aeruginosa phage PaP1, to study DNA replication upon encountering an abasic site lesion. Gp90 exo- can incorporate dNTPs opposite the abasic site, but extension past this site is extremely slow. Among the four dNTPs, dATP is preferentially incorporated opposite the abasic site, consistent with the A-rule. The incorporation is independent of the identity of the nucleotide 5' of the abasic site. The incorporation of dATP opposite the abasic site occurs by direct incorporation of dNTP opposite the abasic site without a -1 frameshift deletion. Extension from an A:abasic site pair by Gp90 exo- is slightly unfavorable relative to those from other abasic site pairs. Incorporation of dATP opposite the abasic site is preferential and shows a biphasic shape, indicating that this incorporation is much faster than the subsequent dissociation of the polymerase from DNA. The template sequence does not affect the dATP incorporation priority, burst amplitude, burst rate, or dATP dissociation constant. Surface plasmon resonance shows that the presence of an abasic site in the template weakens the binding affinity of Gp90 exo- to DNA in a binary or ternary complex in the presence of any one kind of dNTP. This study reveals that Gp90 exo- preferentially inserts A opposite an abasic site via the A-rule, like other DNA polymerases (e.g., Pol θ, KlenTaq, KF exo-, Pols α, δ/PCNA, and Thermococcus litoralis Pol Vent (exo-)), providing further insight into DNA replication mediated by P. aeruginosa phage PaP1 upon encountering an abasic site lesion.