Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity

Scientific Reports
Chih-Hao LuHung-Wen Li

Abstract

RecA plays central roles in the homologous recombination to repair double-stranded DNA break damage in E. coli. A previously identified recA strain surviving high doses of UV radiation includes a dominant RecA E38K mutation. Using single-molecule experiments, we showed that the RecA E38K variant protein assembles nucleoprotein filaments more rapidly than the wild-type RecA. We also used a single-molecule fluorescence resonance energy transfer (smFRET) experiment to compare the nucleation cluster dynamics of wild-type RecA and RecA E38K mutants on various short ssDNA substrates. At shorter ssDNA, nucleation clusters of RecA E38K form dynamically, while only few were seen in wild-type RecA. RecA E38K also forms stable nuclei by specifically lowering the dissociation rate constant, k d . These observations provide evidence that greater nuclei stability and higher ssDNA binding affinity contribute to the observed enhanced recombination activity of the RecA E38K mutant. Given that assembly of RecA nucleoprotein filaments is the first committed step in recombinational repair processes, enhancement at this step gives rise to a more efficient recombinase.

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Citations

Aug 1, 2020·Nucleic Acids Research·Hao-Yen ChangPeter Chi
Jun 20, 2020·Microbiology and Molecular Biology Reviews : MMBR·Shingo Fujii, Robert P Fuchs
Oct 10, 2018·Proceedings of the National Academy of Sciences of the United States of America·Chih-Hao LuHung-Wen Li

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Methods Mentioned

BETA
fluorescence
electrophoresis
fluorescence resonance
FRET

Software Mentioned

Labview
smFRET
Matlab

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