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Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis
J R Carmical1, A Kowalczyk, Y Zou
1Department of Preventive Medicine and Community Health, the Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555, USA.
Abstract:
To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.
Insights
Butadiene diepoxide creates DNA cross-links that cause mutations and block replication in E. coli. These lesions are poorly repaired by the nucleotide excision repair system, indicating a potential health risk.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Butadiene diepoxide is a known mutagenic agent.
- N(2)-N(2) guanine intrastrand cross-links are specific DNA adducts formed by butadiene diepoxide.
- Understanding the mutagenic spectrum and repair of these cross-links is crucial for risk assessment.
Purpose of the Study:
- To identify the mutagenic spectrum of butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links.
- To investigate the impact of these cross-links on DNA replication and repair in Escherichia coli.
Main Methods:
- Synthesis of site-specifically adducted oligodeoxynucleotides containing the cross-links within the human ras 12 codon.
- Transfection of modified DNA into E. coli and assessment of plaque-forming ability and mutation frequency.
- In vitro replication assays using major E. coli polymerases.
- In vitro nucleotide excision repair assays using the E. coli UvrABC complex.
Main Results:
- Both stereoisomeric forms (R,R and S,S) of the DNA cross-links severely decreased plaque-forming ability in E. coli.
- A significant increase in single base substitutions and deletions was observed, with the S,S isomer being more mutagenic.
- Replication of DNA templates containing these cross-links was blocked by major E. coli polymerases.
- The E. coli UvrABC complex showed low efficiency in incising substrates with these intrastrand cross-links.
Conclusions:
- Butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links are highly mutagenic and impede DNA replication.
- These lesions are likely inefficiently repaired by the nucleotide excision repair system in E. coli.
- The findings suggest potential genotoxicity and health risks associated with butadiene exposure.