Abstract
Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.
Highlights
Mutations in the human RAD30A gene, the counterpart of the yeast RAD30, cause the variant form of xeroderma pigmentosum [9, 10]
Translesion DNA Synthesis by pol on the (R)- and (S)-BDO and (R,R)- and (S,S)-BDE N2-Guanine-adducted DNA Substrates—The structures of the BDO and BDE N2-guanine stereoisomers, which were examined in this study, are shown in reduced in vivo, and the presence of these lesions in DNA is a complete block to synthesis by E. coli pol I, II, and III in vitro [18]
Primer extension reactions were carried out to test the ability of yeast pol to perform translesion DNA synthesis on the BDO and BDE N2-guanine-adducted DNA substrates (Fig. 2A)
Summary
Mutations in the human RAD30A gene, the counterpart of the yeast RAD30, cause the variant form of xeroderma pigmentosum [9, 10]. Xeroderma pigmentosum variant cells are hypermutable in response to UV irradiation, and they exhibit a significantly reduced ability to bypass a T-T dimer (reviewed in Ref. 3). Yeast and human pol both efficiently bypass the 7,8-dihydro-8-oxoguanine lesion. Pol is unique among DNA polymerases in its ability to bypass a T-T dimer and a 7,8dihydro-8-oxoguanine lesion efficiently and accurately. We examined the ability of yeast pol to carry out translesion synthesis on DNA substrates containing N2-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Butadiene-mediated carcinogenesis is initiated through its reactive metabolites: butadiene monoepoxide, butadiene diepoxide, and butadiene diolepoxide. We examine the action of yeast pol on these two types of the N2-guanine epoxide-containing DNA substrates
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