The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination

Chien-Der Lee,Ting-Fang Wang

doi:10.1186/1423-0127-16-37

Abstract

Escherichia coli RecA mediates homologous recombination, a process essential to maintaining genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be required for monomer-monomer interaction. Here we report that it has two other distinct roles in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPγS, the NTD is required to capture donor dsDNA. Our results also suggest that the second function of NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic nucleoprotein filament interacts with donor dsDNA.

Highlights

Escherichia coli RecA is the founding member of the RecA protein family
The crystal structure of the RecA-single-stranded DNA (ssDNA) presynaptic filament reveals that N-terminal domain (NTD) are located at the exterior surface of the helical filament
In the RadA right-handed helical filament with 6 monomers per helical pitch, L1 resides near the axis filament and the NTD is located at the exterior surface of the filament

Summary

Introduction

Escherichia coli RecA is the founding member of the RecA protein family. It is essential for the initiation of repair of DNA breaks via homologous recombination, induction of the DNA damage-induced 'SOS' response, and activation of translesion DNA synthesis, as well as development and transmission of antibiotic resistance genes [1,2]. Most known functions of RecA require the formation of a presynaptic helical filament comprised of single-stranded DNA (ssDNA) bound to multiple RecA monomers with ATP. During homologous recombination, this activated form of the helical filament is capable of interacting with homologous double-stranded DNA (dsDNA) to form a heteroduplex DNA molecule. The DNA strands are exchanged, resulting in the displacement of one of the original duplex strands and the subsequent creation of a new heteroduplex (or D-loop). This function is evolutionarily conserved in other members of the RecA family, including archaeal RadA and the eukaryotic proteins, Rad and Dmc

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