RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens

Area of Science:

• Molecular Biology
• Genetics
• Plant Science

Background:

• RAD51 proteins are essential for homologous recombination (HR) and DNA repair.
• Physcomitrium patens exhibits unique high HR rates and gene targeting efficiency in plants.
• RAD51 paralogs regulate and promote RAD51 functions in DNA repair pathways.

Purpose of the Study:

• To elucidate the specific roles of RAD51 and its paralogs in DNA double-strand break (DSB) repair in Physcomitrium patens.
• To investigate the impact of mutating RAD51 and RAD51B genes on DSB repair efficiency and DNA integrity.

Main Methods:

• Construction and analysis of knockout lines: Pprad51-1-2 (mutated in both RAD51-1 and RAD51-2) and Pprad51B (mutated in RAD51B).
• Assessment of hypersensitivity to bleomycin, a DNA-damaging agent.
• Measurement of DNA double-strand break repair kinetics and efficiency.
• Analysis of 5S and 18S rDNA copy number in knockout lines.

Main Results:

• Both Pprad51-1-2 and Pprad51B lines showed hypersensitivity to bleomycin.
• DSB repair was faster than wild type in Pprad51-1-2, but slow in Pprad51B, especially in the second repair phase.
• Absence of RAD51 (in Pprad51-1-2) shifted DSB repair to the non-homologous end joining (NHEJ) pathway, reducing rDNA copy number.
• RAD51B mutation (in Pprad51B) impaired DSB repair kinetics, suggesting a role in damage recognition and HR orchestration.

Conclusions:

• PpRAD51-1 and PpRAD51-2 are functional homologs of ancestral RAD51, critical for homology search in HR.
• RAD51B plays a distinct role in DNA damage response, influencing repair pathway choice and kinetics.
• The study highlights the specialized roles of RAD51 paralogs in maintaining genome stability and HR efficiency in Physcomitrium patens.