Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

Abstract

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the singlemolecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model.

Stretching chimeric DNA: A test for the putative S-form

“…The resulting 'overstretched' form of the molecule is approximately 1.7 times longer than helical B-DNA. Overstretching is of crucial importance for the biological function of DNA: the bacterial protein RecA elongates DNA by a factor of 1.5 upon binding [3,4,5], a mechanism central to homologous recombination and to chromosomal segregation during cell division [6]. However, the nature of the overstretched state remains a source of considerable controversy: some think overstretched DNA in vitro to be a hybridized form called S-DNA (the 'Bto-S' picture) [7,8,9,10,11,12], while a competing picture considers overstretching to signal a conversion to unhybrizided single strands (the 'force-melting' picture) [13,14,15,16,17,18].…”

On the Mechanism of Homology Search by RecA Protein Filaments

“…Biophysical Journal 00(00) [1][2][3][4][5][6][7][8][9][10][11][12] These equations can be solved, producing from Eq. (7)…”

“…During this process a nucleotide sequence of another identical or similar DNA duplex is used for restoring the original sequence in the broken DNA molecule (8,9). Furthermore, the homologous recombination plays a fundamental role in the process of genetic diversity and reassembling of genetic information, which is important for living systems for adaption to changing environments (1,3,5,8,(11)(12)(13).…”

“…In bacteria, a central role in the homologous recombination is played by RecA proteins (11,14,15). They belong to a class of recombination proteins that are conserved across different organisms, from bacteria to mammals (RadA in archea, and Rad51 and Dmc1 in eukaryotes) (3).…”

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

“…Recent work by Fulconis et al 63 utilized the changes in DNA length upon RecA coating to monitor strand-exchange reactions in real time. The authors used magnetic tweezers to stretch RecA-coated single-stranded DNA and monitored the exchange with a strand of a duplex DNA molecule.…”