Specificity of DNA Lesion Bypass by the Yeast DNA Polymerase η

Abstract

DNA polymerase (Pol, xeroderma pigmentosum variant, or Rad30) plays an important role in an errorfree response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thyminethymine cis-syn-cyclobutane dimer. We have purified the yeast Pol and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of ϳ100: 56:14, respectively. The yeast Pol efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol following the Pol action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol␣ was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol may also play a role in bypassing additional DNA lesions, some of which can be error-prone.

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

“…On the other hand, pol η catalyzes significant error-prone nucleotide incorporation opposite a variety of different types of DNA modifications such as (+)-transanti-benzo [a]pyrene-N 2 -dG and an AP site [47]. Interestingly, pol η is able, with relative efficiency, to bypass all the exocyclic adducts tested so far.…”

“…The lesion bypass through combined actions of two polymerases is being studied in order to examine whether such terminated synthesis at the adduct site can be further extended by another polymerase. Two of the Y family DNA polymerases, pol ξ (Rev3 and Rev7) and pol κ, are known to have such elongating function [29,47,51].…”

Somatic immunoglobulin hypermutation

“…In fact, recent evidence suggests that these polymerases can act in concert, where either η or ι inserts an incorrect base, and the ubiquitous polymerase ζ extends from the mismatched pair [40,41], thus stabilizing the mutation. Given the recent data suggesting a role for DSBs in Ig hypermutation, the finding in yeast that increased incidences of base-substitutions near DSBs are entirely dependent on DNA polymerase ζ [42] …”

Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

“…Other investigators using yeast-based assays and/or the enzyme from yeast observed accurate and efficient bypass of 8-oxoG by pol (38), whereby C, A, or G was incorporated opposite 8-oxoG with a relative efficiency of 100:56:14 (39). Moreover, pol binds dCTP opposite both G and 8-oxoG with similar affinities and inserts the correct nucleotide opposite both G and 8-oxoG with similar rates (40).…”