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Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector
1State University of New York at Stony Brook, Department of Pharmacological Sciences 11794-8651.
Abstract:
Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone. Lesions were positioned on opposite strands of the vector. When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion. Mutagenesis occurred in the absence of SOS induction. Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure. For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed. dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector. We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells. Repair DNA synthesis may be involved in this process.
Insights
Abasic sites in DNA are highly mutagenic in E. coli and COS cells, causing mutations even without SOS induction. These DNA lesions may be repaired through DNA synthesis.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Abasic sites, lacking a base, are common DNA lesions that can lead to mutations.
- Understanding mutagenesis at abasic sites is crucial for comprehending DNA stability and repair mechanisms.
Purpose of the Study:
- To investigate mutagenesis occurring at abasic sites within a duplex shuttle vector in bacterial (E. coli) and mammalian (COS) cells.
- To analyze the types and frequencies of mutations induced by different abasic site analogs.
Main Methods:
- Utilized a duplex shuttle vector with synthetic deoxyribose analogs to mimic abasic sites.
- Introduced lesions on opposite strands and analyzed mutational spectra following replication in E. coli and COS cells.
- Examined nucleotide incorporation opposite abasic sites in a gapped duplex vector.
Main Results:
- Bistrand abasic sites, particularly the tetrahydrofuranyl analog, induced significant AT or TA base substitutions (65-80%).
- Mutagenesis occurred independently of the SOS response.
- Propanyl and ethanyl analogs lacking ring structures primarily resulted in single base deletions (>80%).
- A small percentage of G/C and C/G substitutions were observed across all analogs (6-10%).
- Deoxyadenosine monophosphate (dAMP) incorporation was favored opposite tetrahydrofuranyl sites in gapped duplex DNA.
Conclusions:
- Bistrand abasic sites are potent mutagens in both prokaryotic and eukaryotic cells.
- The observed mutagenesis suggests a role for error-prone repair DNA synthesis in processing these lesions.
- Different abasic site structures influence the type and frequency of mutations generated.