Sequence-dependent Induction of Base Pair Substitutions and Frameshifts by Propanodeoxyguanosine during in Vitro DNA Replication

Abstract

Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (CPdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10 3 -10 4 -fold compared with unmodified template primers. Partial blockade was observed 1 base 3 to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the V max /K m values for incorporation of dGMP were similar in both sequence contexts, whereas the V max /K m for dAMP incorporation increased 4.7-fold when the base pair 3 to PdG was changed from C:G to T:A. Oligonucleotides containing 1-and 2-base deletions were major products of replication in both sequence contexts. Fulllength products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation of dAMP opposite PdG. The observation of PdG 3 T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter.

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.…”

Evaluation of the Mutagenic Potential of the Principal DNA Adduct of Acrolein

“…In Vitro DNA Lesion Bypass Assays-DNA polymerase assays were performed essentially as described (24). Primers were 5Ј-radiolabeled using T4 polynucleotide kinase and [␥-…”

Replication of Template-Primers Containing Propanodeoxyguanosine by DNA Polymerase β

“…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). Likewise, PdG induces frameshift and base pair substitution mutations during in vitro replication of adducted template-primers (20,21).The outcome of replication of DNA molecules containing PdG is highly sequence context dependent (20). Using the Klenow fragment (exo ϩ or exo Ϫ ) of DNA polymerase I, we have demonstrated that the identity of the base pair 3Ј to PdG in the template strand influences the choice of dNMP incorporated opposite PdG (20).…”

“…More important, is the identity of the base 5Ј to PdG in the template strand, which determines the outcome of synthesis beyond PdG (20). When the 5Ј base can hydrogen bond to the base opposite PdG at the primer terminus, slippage and extension occur to generate one-base deletions at the site of the PdG residue (20,21). When the 5Ј base is T and the primer terminus contains A, the Klenow fragment extends the primer to a full-length product (20).…”