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Updated: Jul 20, 2026
06:51
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
RecA-ssDNA interaction: induced strand cleavage by hydroxyl radical at a defined distance from the 5' end
E Akaboshi1, P Howard-Flanders
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Molecular & General Genetics : MGG
|February 1, 1990
Summary
RecA protein binds to single-stranded DNA (ssDNA), directing hydroxyl radical cleavage at specific sites. This DNA structural alteration is crucial for homologous pairing and strand exchange.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- RecA protein is essential for DNA repair and recombination.
- RecA protein facilitates homologous pairing and strand exchange.
- The structural dynamics of RecA-ssDNA interactions are not fully understood.
Purpose of the Study:
- To investigate the structural changes induced by RecA protein on single-stranded DNA (ssDNA).
- To determine the precise location of RecA-mediated DNA modifications.
- To elucidate the role of these modifications in DNA recombination processes.
Main Methods:
- Hydroxyl radical footprinting to probe DNA structure.
- Analysis of DNA cleavage patterns induced by hydroxyl radicals in the presence of RecA protein.
- Site-directed mutagenesis of RecA protein and modification of reaction conditions (ATP analogs, Mg2+ concentration).
Main Results:
- RecA protein directs hydroxyl radical cleavage of ssDNA at specific sites, primarily the 11th nucleotide.
- Cleavage patterns suggest RecA induces structural alterations, potentially bending, in ssDNA.
- Cleavage specificity is reduced by ATP-gamma-S, RecA1 mutant protein, or increased Mg2+ concentration.
- Specific cleavage occurs under optimal conditions for RecA filament formation, homologous pairing, and strand exchange.
Conclusions:
- RecA protein induces specific structural alterations on ssDNA.
- These alterations, likely involving DNA bending, are important for RecA's function in homologous pairing and strand exchange.
- The precise mechanism of RecA-ssDNA interaction influences cleavage specificity and recombination efficiency.
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