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General method for quantifying base adducts in specific mammalian genes
D C Thomas1, A G Morton, V A Bohr
1Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
Summary
A new method quantifies DNA adducts in mammalian genomes using ABC excinuclease. This technique measures DNA adduct formation and removal in specific genomic sequences.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA adducts are critical markers of DNA damage and repair.
- Quantifying DNA adducts in specific genomic regions is essential for understanding DNA repair mechanisms.
Purpose of the Study:
- To develop a general method for measuring DNA adduct formation and removal in defined mammalian genomic sequences.
Main Methods:
- Genomic DNA is digested and treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease).
- Alkaline gel electrophoresis and Southern hybridization are used to probe specific sequences.
- ABC excinuclease incises adducted DNA, reducing fragment intensity proportionally to adduct number.
Main Results:
- The method allows for the quantification of DNA adducts in specific genomic sequences.
- The intensity reduction in Southern hybridization directly correlates with the number of DNA adducts.
Conclusions:
- This method provides a versatile tool for studying DNA adducts across various genomic locations.
- The wide substrate range of ABC excinuclease enables the quantification of diverse DNA adducts.
