RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

Abstract

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

“…However, homologs of Mek1 and Hed1 have not been identified, nor has the mechanism by which RAD51 is downregulated been described in organisms other than budding yeast. We recently confirmed that Arabidopsis RAD54 is essential for RAD51-mediated recombination and that this includes meiotic DSB repair in dmc1 mutant plants [74] (Figure 8C and E). This study thus further emphasized that the RAD51-RAD54 pathway is down-regulated or plays a minor role in wild-type Arabidopsis meiosis.…”

“…We note however that asymmetric loading has also been previously suggested (in particular in Arabidopsis) and is thus also possible [19]. The nucleofilament is then the active molecular species and data from budding yeast, mouse and Arabidopsis have demonstrated that DMC1 is the active recombinase during meiosis with RAD51 being an essential co-factor for DMC1, but its strand exchange activity being inhibited [21, 38–40, 46, 47, 57, 74] (Figure 8A). Thus, absence of RAD51 leads to strong defects in DMC1 loading and therefore defects in meiotic DSB repair [19, 38, 58, 75, 76] (Figure 8B).…”

Enhancing HR Frequency for Precise Genome Editing in Plants

“…Effort has also been made in analyzing the effects of modifying the homology-direct repair (HDR) pathway in plants to improve HR-mediated GT ( Lieberman-Lazarovich and Levy, 2011 ; Pradillo et al, 2014 ; Steinert et al, 2016 ; Hernandez Sanchez-Rebato et al, 2021 ). In yeast, the core components of the HR machinery are RAD51, RAD52, and RAD54 ( Li and Heyer, 2008 ).…”

Synthetic Lethality Targeting Polθ

“…Another essential cofactor RAD54 regulates RAD51 activity by stabilizing the RAD51 nucleofilament and remodeling of nucleosomes. It also stimulates the homology search and strand invasion activity of RAD51 [ 27 ].…”

“…Following annealing, the ERCC1 (Excision Repair Cross Complementation Group 1) interacts with XPF (Xeroderma Pigmentosum, Complementation Group F), creating a complex that removes the flanking unannealed non-complementary 3′ssDNA. Finally, DNA Ligase I (LIG1) ligates the paired DNA ends [ 27 , 30 ].…”