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MutS recognition of exocyclic DNA adducts that are endogenous products of lipid oxidation
K A Johnson1, M L Mierzwa, S P Fink
1A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Abstract:
The ability of the methyl-directed mismatch repair system to recognize and repair the exocyclic adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G), the major adduct derived from the endogenous mutagen malondialdehyde, has been assessed both in vivo and in vitro. Both adducts were site-specifically incorporated into M13MB102 DNA, and the adducted genomes were electroporated into wild-type or mutS-deficient Escherichia coli strains. A decrease in mutations caused by both adducts was observed in mutS-deficient strains, suggesting that MutS was binding to the adducts and blocking repair by nucleotide excision repair. This hypothesis was supported by the differences in mutation frequency observed when hemimethylated genomes containing PdG on the (-)-strand were electroporated into a uvrA(-) strain. The ability of purified MutS to bind to PdG- or M(1)G-containing 31-mer duplexes in vitro was assessed using both surface plasmon resonance and gel shift assays. MutS bound to M(1)G:T-containing duplexes with similar affinity to a G:T mismatch but less strongly to M(1)G:C- and PdG-containing duplexes. Dissociation from each of the adduct-containing duplexes occurred at a faster rate than from a G:T mismatch. The present results indicate that MutS can bind to exocyclic adducts resulting from endogenous DNA damage and trigger their removal by mismatch repair or protect them from removal by nucleotide excision repair.
Insights
The methyl-directed mismatch repair system
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Toxicology
Background:
- Endogenous mutagens like malondialdehyde can form DNA adducts.
- Propanodeoxyguanosine (PdG) and M(1)G are major adducts from malondialdehyde.
- DNA repair systems are crucial for maintaining genome integrity.
Purpose of the Study:
- To investigate the methyl-directed mismatch repair system's recognition and repair of PdG and M(1)G adducts.
- To determine the role of MutS in the repair or tolerance of these adducts.
- To assess the binding affinity of MutS to DNA containing these adducts.
Main Methods:
- Site-specific incorporation of PdG and M(1)G adducts into M13MB102 DNA.
- Electroporation of adducted genomes into wild-type and mutS-deficient Escherichia coli strains.
- In vitro binding assays (surface plasmon resonance, gel shift) using purified MutS and adducted DNA duplexes.
Main Results:
- Mutations from both adducts decreased in mutS-deficient strains, suggesting MutS binding and blockage of nucleotide excision repair.
- Differences in mutation frequency in uvrA(-) strains supported the role of MutS.
- Purified MutS exhibited binding to M(1)G:T duplexes, with weaker binding to M(1)G:C and PdG duplexes.
- Dissociation rates from adduct-containing duplexes were faster than from G:T mismatches.
Conclusions:
- MutS can bind to exocyclic DNA adducts formed by endogenous mutagens.
- MutS binding can either trigger mismatch repair or protect adducts from nucleotide excision repair.
- These findings shed light on the cellular response to endogenous DNA damage.
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