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Mutational analysis on structure-function relationship of a holliday junction specific endonuclease RuvC
K Ichiyanagi1, H Iwasaki, T Hishida
1Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|November 13, 1998
Summary
Random mutagenesis identified key residues in Escherichia coli RuvC protein, essential for Holliday junction resolution during homologous recombination. Dimer formation and specific interactions are critical for RuvC function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Escherichia coli RuvC protein is a crucial endonuclease for resolving Holliday junctions in homologous recombination.
- RuvC function involves dimerization, DNA binding, and endonucleolytic cleavage, with its crystal structure previously determined.
Purpose of the Study:
- To identify functionally critical residues of RuvC through random mutagenesis.
- To elucidate the roles of specific residues in RuvC's catalytic activity, dimerization, and DNA binding.
Main Methods:
- Random mutagenesis of ruvC genes.
- In vivo and in vitro characterization of mutant RuvC proteins.
- Analysis of dimerization, DNA binding, and cleavage activities.
Main Results:
- Mutations frequently occurred at the four acidic residues of the catalytic center.
- Several mutants affected the dimer interface; one failed to dimerize, while others dimerized but were cleavage-defective.
- F69L and K118R mutants could not cleave junctions despite dimer formation and DNA binding.
Conclusions:
- Random mutagenesis revealed structurally and functionally important, conserved RuvC residues.
- Dimer formation and intact subunit interactions are vital for RuvC's junction binding and cleavage.
- Phenylalanine-69 and Lysine-118 are critical for junction cleavage activity.