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Single-molecule studies reveal a traveling wave-like model for homologous recombination

Date:12-08-2020 Print

Homologous recombination (HR) is a major pathway for repair of double-stranded DNA break. RecA polymerizes along single-stranded DNA at the end of a broken DNA to form a presynaptic filament, which is a right-handed helix with ~6 monomers per turn and 3 bases per monomer. The presynaptic filament catalyzes search for homologous sequences and promotes strand exchange with a donor DNA. HR has been known for decades, but the underlying molecular mechanism remained unclear. By using single molecular magnetic tweezers (MT) and fluorescent resonant energy transfer (FRET), the Li Ming Lab of the Institute of Physics, Chinese Academy of Sciences, managed to catch transient states in HR and built a traveling model for RecA-mediated HR.

In an effort to solve the challenge to catch the transient states, the researchers realized that the question of how strand exchange progresses is actually equivalent to how it is blocked. They therefore designed a series of mismatch-containing donor DNAs to assess the dynamics of HR at the single-molecule level. They found that even though 3 base pairs (bp) is still the basic unit as previously reported, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ~9 bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ~9 bp before the match-mismatch joint.

The results suggest that the homology checking and the strand exchange are separated in space, with the strand exchange lagging behind the homology checking by ~9 bp. In order to explain their observations, the researchers made a hypothesis that there exists a transitional segment between the homology-checking region and the post strand-exchanged region. Accordingly, they built a model of strand exchange: the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in ~9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post strand exchanged duplex (Fig. 2).

Fig. 1. MT assay of the strand exchange. (Image by Institute of Physics)

Fig. 2. Stepwise progression in HR. (Image by Institute of Physics)

This study entitled "Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination" was published on PNAS.

This work was supported by the National Natural Science Foundation of China, the CAS Key Research Program of Frontier Sciences, the Youth Innovation Promotion Association of CAS and the K. C. Wong Education Foundation.

Institute of Physics
Ming Li

Key word:
homologous recombination | RecA | strand exchange | molecular mechanism | dynamic configuration

Using single molecule techniques and mismatch sequences, a new dynamic model of homologous recognition and strand exchange process during homologous recombination mediated by RecA was built.