Species-specific coevolution of RecA-RecN interfaces governs DNA double-strand break repair in Escherichia coli

DNA double-strand breaks (DSBs) threaten genome stability and cell survival but can be faithfully repaired through homologous recombination (HR). RecN, a bacterial protein closely related to the structural maintenance of chromosomes family, cooperates with RecA in HR-dependent DSB repair, yet the molecular basis and physiological relevance of their interaction remain unclear. Here, we investigated the functional interplay between RecA and RecN during DSB repair by heterologously expressing Pseudomonas aeruginosa RecA (paRecA) and RecN (paRecN) in Escherichia coli. We found that in E. coli ∆recA ∆recN cells, co-expression of paRecA and paRecN fully restored MMC resistance, whereas co-expression of E. coli RecA (ecRecA) with paRecN conferred partial resistance to mitomycin C (MMC), demonstrating species-specific compatibility. Expression analysis revealed that paRecN was poorly expressed in E. coli, but codon optimization significantly enhanced its abundance and repair activity. We further identified gain-of-function paRecN mutants (I73T and R453H) that restored repair without increased expression. These mutants displayed species-specific adaptation, which improved compatibility with ecRecA but reduced functionality with paRecA. Fluorescence microscopy revealed that MMC-induced nucleoid localization was increased in paRecNI73T and paRecNR453H compared with paRecN. Collectively, these findings demonstrate that coevolution optimizes the RecA-RecN interface to ensure efficient DSB repair.