Abstract
DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) 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 approximately 100:56:14, respectively. The yeast Pol(eta) 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(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) 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(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.
Highlights
During replication, a variety of DNA lesions can block replicative DNA polymerases
DNA polymerase activity of the yeast Pol requires Mg2ϩ, and the polymerase was active with 1–20 mM MgCl2 and 0 – 80 mM KCl
The recently described DNA Pol is encoded by the RAD30 gene in yeast and the xeroderma pigmentosum variant (XPV) gene in humans [17, 18, 22, 24]
Summary
Polymerase; AP, apurinic/apyrimidinic; XPV, xeroderma pigmentosum variant; AAAF, N-acetoxy-N-2acetylaminofluorene; AAF, acetylaminofluorene; FPLC, fast protein liquid chromatography; nt, nucleotide; 8-oxoG, 8-oxoguanine. Response of the yeast Pol to several other DNA lesions in vitro. We (i) show the properties of the yeast Pol in response to an 8-oxoguanine, an AAF-modified guanine, and an AP site in the DNA template; and (ii) provide evidence that Pol can be error-prone during DNA synthesis opposite some lesions
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