Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion

Abstract

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn2+-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn2+ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn2+-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn2+ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.

Structural and Biochemical Analysis of the Recombination Mediator Protein RecR from Campylobacter jejuni

“…Consistently, cjRecR displayed substantial binding to ssDNA, whereas drRecR and csRecR did not ( Figure 3 A) [ 8 , 9 , 12 ]. Given that Campylobacterota species do not express RecF, which contributes to DNA recognition, RecR proteins from RecF-deficient Campylobacterota bacteria seem to have evolved the ability to bind DNA more efficiently than RecR proteins from RecF-expressing bacteria [ 5 , 6 ].…”

“…cjRecO protein was produced in E. coli cells and purified by affinity and ion exchange chromatography as described previously [ 6 ].…”

“…As a result, RecR directs RecO to the ssDNA side through specific binding to RecO and is expected to recruit RecA on SSB-coated ssDNA [ 3 , 4 ]. However, this model is difficult to generalize to all bacterial species, given that some bacterial species, including Campylobacter jejuni and Helicobacter pylori , do not have the recF gene and employ the RecF-independent RecOR pathway for homologous recombination [ 5 , 6 ]. Moreover, the RecOR pathway has been shown to coexist with the RecFOR pathway even in RecF-expressing bacteria [ 7 ].…”

“…However, these features have been revealed in RecF-expressing bacteria from the phyla Deinococcota, Actinomycetota, and Pseudomonadota and have not been characterized in RecF-deficient bacteria. RecR from RecF-deficient bacteria is expected to display both common and unique features compared to that from RecF-expressing bacteria, as shown for RecO [ 6 , 13 ]. RecO from RecF-deficient Campylobacter jejuni , which belongs to the phylum Campylobacterota and causes food poisoning in humans, presents a curved three-domain structure, as shown for other RecO orthologs [ 6 ].…”

“…RecR from RecF-deficient bacteria is expected to display both common and unique features compared to that from RecF-expressing bacteria, as shown for RecO [ 6 , 13 ]. RecO from RecF-deficient Campylobacter jejuni , which belongs to the phylum Campylobacterota and causes food poisoning in humans, presents a curved three-domain structure, as shown for other RecO orthologs [ 6 ]. However, C. jejuni RecO (cjRecO) coordinates a zinc ion using a unique sequence and has substantially lower ssDNA-binding affinity than SSB and other RecO proteins.…”