Replication of Template-Primers Containing Propanodeoxyguanosine by DNA Polymerase β

Abstract

Propanodeoxyguanosine (PdG) is a model for several unstable exocyclic adducts formed by reaction of DNA with bifunctional carbonyl compounds generated by lipid peroxidation. The effect of PdG on DNA synthesis by human DNA polymerase ␤ was evaluated using template-primers containing PdG at defined sites. DNA synthesis was conducted in vitro and the products were analyzed by polyacrylamide gel electrophoresis and DNA sequencing. The extent of PdG bypass was low and the products comprised a mixture of base pair substitutions and deletions. Sequence analysis of all of the products indicated that the deoxynucleoside monophosphate incorporated "opposite" PdG was complementary to the base 5 to PdG in the template strand. These findings are very similar to recent results of Efrati et al. (Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1997) J. Biol. Chem. 272, 2559 -2569) obtained in DNA replication of template-primers containing abasic sites and suggest that PdG is a non-informational lesion when acted upon by polymerase (pol) ␤. In addition to base pair substitutions and one-or two-base deletions, a four-base deletion was observed and the mechanism of its formation was probed by site-specific mutagenesis. The results indicated that this deletion occurred by one-base insertion followed by slippage to form a four-base loop followed by extension. All of the observations on pol ␤ replication of PdG-containing template-primers are consistent with a mechanism of lesion bypass that involves template slippage and dNTP stabilization followed by deoxynucleoside monophosphate incorporation and extension. This mechanism of PdG bypass is completely different than that previously determined for the Klenow fragment of DNA polymerase I and is consistent with recent structural models for DNA synthesis by pol ␤.Mutations are the cause of genetic disease and arise during the copying of normal or damaged DNA templates. The factors that determine the frequency and types of mutations include the identity of the DNA polymerase, the composition of the deoxynucleotide pool, the local sequence context of the template, and, in the case of damaged templates, the structure of the DNA lesion (1-16). Our laboratory and others have investigated the effect of propanodeoxyguanosine (PdG) 1 on the fidelity of DNA replication in vivo and in vitro (17)(18)(19)(20)(21)(22). PdG has been used as a model with which to assess the biological effects of several unstable DNA adducts derived from the reaction of dG residues with bifunctional aldehydes (23, 24). PdG is a relatively small lesion that completely blocks Watson-Crick base pairing but induces little distortion in the local structure of DNA molecules to which it is introduced (25, 26). Transformation of recombinant viral genomes or shuttle vectors containing site-specifically positioned PdG residues into bacterial and mammalian cells demonstrates that PdG is highly mutagenic and capable of inducing a range of base pair substitutions and frameshift mutations in vivo (17)(18)(19)22...

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II

“…M 1 dG adducts, produced during lipid peroxidation, seem to be repaired by NER in both bacteria and human cells (23). Like other adducts, such as CPDs, which arrest RNAPs and are repaired by TCR, PdG, the stable analog of M 1 dG, also inhibits replication by DNA polymerases (27,28,37). Therefore, it was reasoned that M 1 dG or PdG in the transcribed strand of active genes might impede translocation by RNAPs and potentially initiate TCR.…”

“…The induction of frameshift mutations by PdG and M 1 dG suggests that the adducts alter DNA polymerase elongation (19,20,22). PdG does, in fact, interfere with nucleotide insertion and translocation by Klenow fragment and DNA polymerase ␤ in vitro (27,28); therefore, it is possible that MDA adducts may also impede RNA polymerases. If so, M 1 dG in expressed genes might be recognized and removed by TCR.…”

The Frameshift Infidelity of Human DNA Polymerase λ

“…3) and the lack of a further increase in rate with increasing run length suggests the involvement of additional mechanisms and/or unusual interactions of Pol with its substrates. Possibilities include misalignment in the polymerase active site (35)(36)(37)(38)(39), nucleotide misinsertion followed by primer relocation (32,40), or some other mechanism. The fact that the relationship between single-base deletion rates and homopolymeric run length are different for Pol compared with Pol ␤ (Fig.…”

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

“…NMR spectroscopy of the PdGadducted oligodeoxynucleotides has revealed that when placed opposite dC, PdG adopts a syn orientation within the duplex and introduces a localized structural perturbation that is pHand sequence-dependent (9,10). The inability of PdG to form normal Watson-Crick hydrogen bonds severely blocks DNA synthesis both in vitro (11,12) and in vivo (13)(14)(15)(16), and the replication that does occur results in mutations (13)(14)(15)(16). Specifically, PdG-induced base substitutions occurred at an overall frequency of 7.8 ϫ 10 Ϫ2 and 7.5 ϫ 10 Ϫ2 /translesion synthesis in the COS-7 (14) and in the nucleotide excision repair-deficient human cells (16), respectively.…”