Abstract
Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.
Highlights
Homologous recombination (HR) is essential for genomic integrity by rescuing collapsed replication forks and repairing DNA double-strand breaks in virtually all organisms [1,2,3,4]
We developed a capillary electrophoresis (CE)-laser-induced fluorescence polarization (LIFP) assay for monitoring RecA nucleofilaments, which was performed on laboratory-built CE-LIFP apparatus (Supplementary Figure S1A–C)
CE separation rapidly removes both unbound and bound ATP away from the RecA-DNA filaments zone. This largely inhibits ATP hydrolysis and avoids a dramatic change in the binding stoichiometry of RecA nucleofilaments caused by ATP hydrolysis-induced end disassembly
Summary
Homologous recombination (HR) is essential for genomic integrity by rescuing collapsed replication forks and repairing DNA double-strand breaks in virtually all organisms [1,2,3,4]. A central feature of HR is a DNA strand exchange reaction catalyzed by RecA and its homologs, which constitute a family of proteins conserved from bacteria to eukaryotes [7, 8]. To initiate DNA strand exchange, Escherichia coli RecA proteins are loaded on replication- or damage-induced. We sought to examine the dynamics, structure and functions of RecA nucleofilaments under physiologically relevant conditions. For this purpose, we developed a novel assay uniquely combining highly efficient capillary electrophoresis (CE) separation with laser-induced fluorescence polarization (LIFP) detection. Because the CE separation is rapid and is carried out under physiological conditions, we could rapidly monitor the physiologically relevant dynamics of RecA-DNA filaments and HR. By combining systematic point mutations of RecA with a bacterial HR reporter assay, we show that the newly identified nucleofilaments are fully compatible with HR in an in vivo setting
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