Translesion Synthesis past Acrolein-derived DNA Adduct, γ-Hydroxypropanodeoxyguanosine, by Yeast and Human DNA Polymerase η

Irina G Minko,M Todd Washington,Manorama Kanuri,Louise Prakash,Satya Prakash,R Stephen Lloyd

doi:10.1074/jbc.m207774200

Irina G Minko, M Todd Washington + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m207774200

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Abstract

gamma-Hydroxy-1,N(2)-propano-2'deoxyguanosine (gamma-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol eta can incorporate a C opposite gamma-HOPdG at approximately 190- and approximately 100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at approximately 8- and approximately 19-fold lower efficiency. Although DNA synthesis past gamma-HOPdG by yeast pol eta was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of approximately 10(-1) to 10(-2). Because gamma-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma-HOPdG. For both yeast and human pol eta, the ring open reduced gamma-HOPdG adduct was less blocking than gamma-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing gamma-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol eta might potentially contribute to both error-free and mutagenic bypass of gamma-HOPdG.

Highlights

Acrolein (Fig. 1), the simplest ␣,␤-unsaturated aldehyde, is an environmental contaminant and a product of inborn metabolism
We show that both yeast and human pol ␩ can incorporate a C opposite ␥-HOPdG at ϳ190- and ϳ100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at ϳ8- and ϳ19-fold lower efficiency
A 21-mer primer was annealed to the template DNA so that it allowed the addition of 9 nucleotides before encountering the adduct (Ϫ9 primer)

Summary

RESULTS

In Vitro Lesion Bypass with Yeast DNA Polymerase ␩—To examine whether yeast pol ␩ was able to replicate past a ␥-HOPdG adduct, running start primer extension experiments were performed (Fig. 2A). To identify the nucleotide that is incorporated by this polymerase opposite the adducted base, single-nucleotide incorporation experiments were carried out using standing start DNA substrates in which 3Ј terminus of the primer was located one nucleotide before the lesions (Ϫ1 primers) (Fig. 2B). Yeast pol ␩ is capable of bypassing the ␥-HOPdG adduct, and in contrast to all other polymerases tested so far [22,23,24], it predominantly incorporates the correct nucleotide opposite and downstream of the lesion These data show that a cyclic PdG is a much stronger block for replication by yeast pol ␩ than an acyclic reduced ␥-HOPdG, but neither of the model.

Fidelity of incorporationb

Base pair substitutions

DISCUSSION