Phosphorylation of RecA at the Loop 1 region modulates its activity in response to DNA damage.

Deinococcus radiodurans exhibits exceptional DNA repair and radiation resistance. Protein phosphorylation, a key regulatory mechanism, modulates amino acid properties by replacing a hydroxyl group with a phosphate group. In this study, we perform phosphoproteomic analyses on D. radiodurans exposed to ionizing radiation. Comparative analysis reveals altered phosphorylation levels in proteins involved in replication, transcription, translation, and free radical scavenging. Notably, DrRecA, a critical protein for homologous recombination repair and the SOS response, exhibits a significant decrease in phosphorylation level by approximately 69.2 % following exposure to ionizing radiation. The phosphorylated residue identified in the current study, Ser174, is located in the highly conserved DNA-interacting Loop 1 region. The side chain of Ser174 itself is crucial for DrRecA's ATPase activity, single-stranded DNA (ssDNA) binding, and strand exchange activity. The phosphorylation-mimicking mutant (S174D) alters its DNA-binding preference toward ssDNA over dsDNA. Despite significantly reduced ATPase activity, the S174D mutant forms elongated RecA-ssDNA filaments and promotes DNA strand exchange reaction in the presence of single-stranded DNA-binding protein. Thus, the altered phosphorylation of DrRecA may play a regulatory role in ssDNA signal sensing, facilitating the homologous recombination in D. radiodurans. These findings provide novel insights into the phosphorylation-mediated stress response in D. radiodurans and highlight the regulatory mechanisms controlling RecA function in recombinational repair and the SOS response.