High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity

TL;DR

This study reveals that RecA protein enables highly sensitive and directional homology recognition during DNA recombination, functioning as a kinetic proofreading mechanism to maintain genetic diversity with high fidelity.

Contribution

The paper provides detailed in vitro analysis showing RecA's ability to detect mismatches and its directional influence, advancing understanding of homology recognition mechanisms.

Findings

RecA detects single mismatches early in recombination.

Mismatch location affects recombination efficiency.

Homology recognition operates via a kinetic proofreading cascade.

Abstract

Homologous recombination plays a key role in generating genetic diversity, while maintaining protein functionality. The mechanisms by which RecA enables a single-stranded segment of DNA to recognize a homologous tract within a whole genome are poorly understood. The scale by which homology recognition takes place is of a few tens of base pairs, after which the quest for homology is over. To study the mechanism of homology recognition, RecA-promoted homologous recombination between short DNA oligomers with different degrees of heterology was studied in vitro, using fluorescence resonant energy transfer. RecA can detect single mismatches at the initial stages of recombination, and the efficiency of recombination is strongly dependent on the location and distribution of mismatches. Mismatches near the 5' end of the incoming strand have a minute effect, whereas mismatches near the 3' end hinder strand exchange dramatically. There is a characteristic DNA length above which the sensitivity to heterology decreases sharply. Experiments with competitor sequences with varying degrees of homology yield information about the process of homology search and synapse lifetime. The exquisite sensitivity to mismatches and the directionality in the exchange process support a mechanism for homology recognition that can be modeled as a kinetic proofreading cascade.