Both DNA methylation and homologous recombination (HR) are extensively studied. In bacteria, Dam methylation is the most studied DNA modification, while RecA-mediated HR is a primary mechanism to repair DNA damages including double-stranded breaks, single-stranded gaps, and stalled replication forks. While HR regulation by proteins is extensively studied, whether methylation of DNA itself directly affects the functions of RecA and HR remains unclear. Mainly by single-molecule experiments, we report that Dam methylation of single-stranded DNA (ssDNA) promotes RecA assembly, partially by reducing the effective charge of ssDNA under counterion screening. Furthermore, Dam methylation of double-stranded DNA promotes homologous pairing, joint molecule growth, and strand exchange. In cellular experiments, dam deletion impairs HR, whereas hypermethylation of the adenines in the genome enhances HR in P1 transduction assays and DNA-damage sensitivity tests without significantly upregulated HR-related genes. In addition, the preference of RecA for Dam-methylated DNA in RecA assembly and homologous pairing is conserved across divergent species covering a gram-negative bacterium Klebsiella pneumoniae, a gram-positive bacterium Bacillus subtilis, and a flowering plant Arabidopsis thaliana. Dam methylation of ssDNA increases the ATPase activity of molecular motors such as RecQ helicase that containing RecA-like domains. These findings reveal effects of DNA methylation and mechanisms regulating RecA-mediated HR and molecular motors.
Read more at PNAS:
https://www.pnas.org/doi/10.1073/pnas.2508652122
Photo captioned: RecA assembles faster on methylated ssDNA. In MT experiments, we used a 6 k-nt ssDNA containing 22 GATC sites. In BLI experiments, we used a 20-nt ssDNA containing only one GATC site. (A) In MT experiments, RecA assembly leads to ssDNA elongation. (B and C) RecA assembly on SSB-coated ssDNA with ATP or ATPγS. (D) RecA assembly on SSB-coated ssDNA with ATP in the presence of RecO and RecR. (E) RecA assembly on naked ssDNA with ATP. (F) RecA nucleation with ATPγS in BLI experiments. (G) Dam methylation reduces electrophoretic mobility of ssDNA. See the original, uncropped gel images in the SI Appendix, Fig. S8. (H) Assembly of RecA E38K on ssDNA in MT experiments. (I) RecA E38K nucleation with ATPγS in BLI experiments. In panels (B to E, and H), shadows represent SE from more than three DNA molecules, and arrows indicate the addition of RecA or other proteins in MT experiments. In panels (F and I), global fitting curves for three RecA concentrations are plotted as black dashed lines.
20 Aug 2025
