The Essential Role of RAD51 DNA Binding Site II in Homologous Recombination Activity in Arabidopsis thaliana

Core Concepts

The recombinogenic activity of RAD51 DNA binding Site II is essential for homologous recombination in Arabidopsis, but not required for meiotic recombination when DMC1 is present.

Abstract

The study investigates the function of RAD51 DNA binding Site II in Arabidopsis thaliana. The authors generated an Arabidopsis RAD51-II3A mutant, where three key amino acids in Site II were mutated to alanine, and characterized its activity in vivo. Key highlights: The RAD51-II3A mutant retains the ability to bind DNA and form nucleoprotein filaments in both somatic and meiotic cells, indicating that Site II is not required for nucleofilament formation. However, the RAD51-II3A mutant is severely defective in DNA repair and homologous recombination in somatic cells, demonstrating that the recombinogenic activity of Site II is essential. Interestingly, the RAD51-II3A mutant fully complements the meiotic defects of the rad51 mutant, suggesting that the recombinogenic activity of RAD51 is not essential for meiotic recombination when the meiosis-specific recombinase DMC1 is present. The RAD51-II3A mutant exhibits a dominant negative effect, disturbing the function of the native RAD51, which is in contrast with the observations in yeast. The results confirm the conservation of the structure and function of RAD51 DNA binding Site II in plants and provide insights into the distinct roles of RAD51 and DMC1 in meiotic recombination.

Stats

Arabidopsis RAD51 displays 54% identity (68% similarity) to S. cerevisiae Rad51 and 68% identity (84% similarity) to human RAD51. The three essential amino acids in RAD51 Site II (R133, R306, K316) are conserved between Arabidopsis, yeast, and human. Arabidopsis RAD51-II3A plants show severe hypersensitivity to the DNA damaging agent Mitomycin C. Somatic homologous recombination is dramatically reduced in Arabidopsis RAD51-II3A plants.

Quotes

"RAD51-II3A has a dominant negative effect and disturbs the function of the native RAD51." "The recombinogenic activity carried by DNA binding Site II is not essential for meiotic DSB repair when DMC1 is present."

Key Insights Distilled From

DNA binding site II is required for RAD51 recombinogenic activity in Arabidopsis thaliana

by Petiot,V., W... at www.biorxiv.org 03-08-2024

https://www.biorxiv.org/content/10.1101/2024.03.07.583932v1

Deeper Inquiries

How do the biochemical properties of RAD51 differ between Arabidopsis, yeast, and human, leading to the observed differences in the dominant negative effects of the RAD51-II3A mutant

In Arabidopsis, yeast, and human, RAD51 proteins play a crucial role in DNA repair through homologous recombination. However, there are subtle biochemical differences in RAD51 proteins among these organisms that lead to variations in their dominant negative effects when mutated. One key difference lies in the DNA binding properties of RAD51. In yeast and human RAD51, the mutation of three specific residues in DNA binding Site II to Alanine results in the separation-of-function mutant Rad51-II3A. This mutant retains the ability to bind single-stranded DNA (ssDNA) and form the nucleoprotein filament but is defective in strand invasion and D-loop formation. Interestingly, in Arabidopsis, the RAD51-II3A mutant also exhibits a dominant negative effect, disrupting the function of the native RAD51 protein. This dominant negative effect is likely due to the increased accumulation of RAD51 at DNA break sites, possibly interfering with the binding of endogenous RAD51 and impeding productive recombination. Therefore, the differences in the biochemical properties of RAD51 proteins, particularly in DNA binding and filament formation, contribute to the observed variations in the dominant negative effects of the RAD51-II3A mutant in Arabidopsis, yeast, and human.

What are the specific mechanisms by which DMC1 downregulates the recombinogenic activity of RAD51 during meiosis in Arabidopsis

During meiosis in Arabidopsis, DMC1 plays a crucial role in downregulating the recombinogenic activity of RAD51. DMC1 and RAD51 are both recombinases involved in meiotic recombination, with DMC1 being the primary recombinase responsible for catalyzing homology search and strand exchange during meiosis. One mechanism by which DMC1 downregulates the activity of RAD51 is through competition for binding to DNA substrates. DMC1 has a higher affinity for DNA substrates compared to RAD51, allowing it to outcompete RAD51 for binding to single-stranded DNA (ssDNA) during meiotic recombination. This preferential binding of DMC1 prevents RAD51 from forming active nucleoprotein filaments and carrying out strand invasion, effectively inhibiting the recombinogenic activity of RAD51. Additionally, recent evidence suggests that DMC1 may directly contribute to the downregulation of RAD51 activity by modulating the biochemical properties of RAD51 at DNA break sites. This regulatory mechanism ensures that DMC1 is the primary recombinase driving meiotic recombination, while RAD51 plays a supporting role in the process.

How do the distinct roles of RAD51 and DMC1 in meiotic recombination contribute to the generation of genetic diversity in plants

The distinct roles of RAD51 and DMC1 in meiotic recombination contribute significantly to the generation of genetic diversity in plants. During meiosis, DMC1 and RAD51 work together to promote the repair of DNA double-strand breaks (DSBs) and facilitate the exchange of genetic material between homologous chromosomes. DMC1, being the primary recombinase in meiotic cells, catalyzes the homology search and strand exchange process, ensuring accurate chromosome segregation and genetic diversity among meiotic products. By downregulating the recombinogenic activity of RAD51, DMC1 ensures that the majority of recombination events during meiosis are driven by DMC1, leading to the formation of crossovers and genetic diversity. RAD51, on the other hand, plays a supporting role in meiotic recombination, assisting DMC1 in the repair of DSBs and promoting the formation of stable nucleoprotein filaments. While RAD51 is not the primary recombinase in meiosis, its presence is essential for efficient DSB repair and the successful completion of meiotic recombination. Overall, the coordinated actions of RAD51 and DMC1 in meiotic recombination ensure the faithful repair of DNA breaks, the exchange of genetic material between homologous chromosomes, and the generation of genetic diversity in plant populations.