Single Molecule Study of the Polymerization of RecA on dsDNA: The Dynamics of Individual Domains

Nitzan Maman; Pramod Kumar; Amarjeet Yadav; Mario Feingold

doi:10.3389/fmolb.2021.609076

Single Molecule Study of the Polymerization of RecA on dsDNA: The Dynamics of Individual Domains

Front Mol Biosci. 2021 Mar 22:8:609076. doi: 10.3389/fmolb.2021.609076. eCollection 2021.

Authors

Nitzan Maman^{1

2}, Pramod Kumar¹, Amarjeet Yadav^{1

3}, Mario Feingold^{1

2}

Affiliations

¹ Department of Physics, Ben Gurion University of the Negev, Beer Sheva, Israel.
² The Ilse Katz Center for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva, Israel.
³ Department of Applied Physics, Babasaheb Bhimrao Ambedkar University, Lucknow, India.

Abstract

In the Escherichia coli, RecA plays a central role in the recombination and repair of the DNA. For homologous recombination, RecA binds to ssDNA forming a nucleoprotein filament. The RecA-ssDNA filament searches for a homologous sequence on a dsDNA and, subsequently, RecA mediates strand exchange between the ssDNA and the dsDNA. In vitro, RecA binds to both ssDNA and dsDNA. Despite a wide range of studies of the polymerization of RecA on dsDNA, both at the single molecule level and by means of biochemical methods, important aspects of this process are still awaiting a better understanding. Specifically, a detailed, quantitative description of the nucleation and growth dynamics of the RecA-dsDNA filaments is still lacking. Here, we use Optical Tweezers together with a single molecule analysis approach to measure the dynamics of the individual RecA domains on dsDNA and the corresponding growth rates for each of their fronts. We focus on the regime where the nucleation and growth rate constants, k _n and k _g , are comparable, leading to a coverage of the dsDNA molecule that consists of a small number of RecA domains. For the case of essentially irreversible binding (using ATPγS instead of ATP), we find that domain growth is highly asymmetric with a ratio of about 10:1 between the fast and slow fronts growth rates.

Keywords: nucleation and growth; optical tweezers; protein-DNA interaction; recA; single molecule.