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Infidelity of DNA synthesis associated with bypass of apurinic sites
Abstract:
The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.
Insights
Apurinic sites, DNA damage locations, can cause mutations. Studies show these sites are mutagenic lesions, with specific enzymes influencing mutation frequency and type.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Apurinic sites are DNA lesions lacking a purine base.
- These sites can arise from spontaneous depurination or DNA damage.
- Their role in mutagenesis requires further investigation.
Purpose of the Study:
- To investigate the mutagenic potential of apurinic sites in vivo.
- To determine the role of different DNA polymerases in copying apurinic sites.
- To elucidate the mechanism of mutagenesis at apurinic sites.
Main Methods:
- Transfection of depurinated phi X174 DNA into SOS-induced Escherichia coli spheroplasts.
- Treatment with apurinic endonuclease to identify mutagenic lesions.
- In vitro DNA synthesis assays using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase.
- Sequence analysis of revertant phage DNA.
Main Results:
- Apurinic sites were confirmed as mutagenic lesions.
- Avian myeloblastosis DNA polymerase copied apurinic sites more frequently than E. coli DNA polymerase I.
- Mutagenesis involved bypass of apurinic sites, with a preference for deoxyadenosine incorporation.
Conclusions:
- Apurinic sites are significant mutagenic lesions in DNA.
- DNA polymerase choice influences mutation frequency and outcomes at apurinic sites.
- Mutagenesis at apurinic sites occurs via lesion bypass during DNA replication.