Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis.
Mutations produced in Escherichia coli by apurinic sites are believed to arise via SOS-assisted translesion replication. Analysis of replication products synthesized on depurinated single-stranded DNA by DNA polymerase III holoenzyme revealed that apurinic sites frequently blocked in vitro replication. Bypass frequency of an apurinic site was estimated to be 10-15%. Direct evidence for replicative bypass was obtained in a complete single-stranded----replicative form replication system containing DNA polymerase III holoenzyme, single-stranded DNA binding protein, DNA polymerase I, and DNa ligase, by demonstrating the sensitivity of fully replicated products to the apurinic endonuclease activity of E. coli exonuclease III. Termination at apurinic sites, like termination at pyrimidine photodimers, involved dissociation of the polymerase from the blocked termini, followed by initiations at available primer templates. When no regular primer templates were available, the polymerase underwent repeated cycles of dissociation and rebinding at the blocked termini and, while bound, carried out multiple polymerization-excision reactions opposite the apurinic sites, leading to turnover of dNTPs into dNMPs. From the in vitro turnover rates, we could predict with striking accuracy the specificity of apurinic site mutagenesis, as determined in vivo in depurinated single-stranded DNA from an M13-lac hybrid phage. This finding is consistent with the view that DNA polymerase III holoenzyme carries out the mutagenic "misinsertion" step during apurinic site mutagenesis in vivo and that the specificity of the process is determined primarily by the polymerase. SOS-induced proteins such as UmuD/C might act as processivity-like factors to stabilize the polymerase-DNA complex, thus increasing the efficiency of the next stage of past-lesion polymerization required to complete the bypass reaction.
Hevroni
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D
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Livneh
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D001080:Apurinic Acid / Q000235:genetics*; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D011119:Polynucleotides / Q000235:genetics*; D011740:Pyrimidine Dimers / Q000378:metabolism; D013014:SOS Response, Genetics
D004277:DNA, Single-Stranded; D009711:Nucleotides; D011119:Polynucleotides; D011740:Pyrimidine Dimers; D001080:Apurinic Acid; D004256:DNA Polymerase I; D004258:DNA Polymerase III