RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

Abstract

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.

POLQ plays a key role in the repair of CRISPR/Cas9‐induced double‐stranded breaks in the moss Physcomitrella patens

“…Mid‐wavelength UV (UV‐B) is biologically relevant to the plants, considering the potential influence of solar UV‐B radiation on the genomic stability of plant populations (Ries et al ., ). It has been shown previously that major components of the HR machinery, such as RAD51 or RAD51 paralogs are essential for resistance to UV‐B in P. patens (Schaefer et al ., ; Charlot et al ., ). Here we show that POLQ is not involved in response to UV‐B in P. patens .…”

“…S1). The PpPOLQ-KO knockout cassette used for the gene knockout experiment bears a 180-bp 5 0 targeting fragment (coordinate 4544À4724 on Pp3c5_12930 in Phytozome) and a 212-bp 3 0 targeting fragment (coordinate 4725À4937 on Pp3c5_12930 in Phytozome) of the POLQ gene, flanking a pAct:: hygroR cassette from the pBHRF plasmid (Charlot et al, 2014). The lig4 mutant was generated via GT using the classical GT technology (Trouiller et al, 2006).…”

Can mosses serve as model organisms for forest research?

“…Though mosses are haploid during a large fraction of their life, i.e., possess just one allele of each gene, they can often cope with higher amounts of toxic or mutagenic substances compared to flowering plants (Charlot et al 2014;Harrison et al 2009). Considering the absence of a second allele which could balance somatic mutations, this drawback is compensated by a high metabolic redundancy (Lang et al 2005), whole genome duplications (Rensing et al 2007), and the arrest of the cell cycle after DNA synthesis in the G2 phase (Schween et al 2003), combined with efficient DNA repair mechanisms such as homologous recombination (Kamisugi et al 2006).…”

“…The molecular basis of the high rate of homologous recombination of the "green yeast" P. patens has been the subject of several studies, identifying some important factors (Charlot et al 2014;Kamisugi et al 2012), although a complete mechanistic model is still lacking.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The ratio between TGR and TGI events was identical, but the proportion of plants with a single copy replacement was significantly higher with CRISPR (40.5%) than without (15%). Classical GT in P. patens was recently described as dependent on the classical RAD51-mediated HR repair pathway [112,113,118]. Interestingly, with CRISPR, HDR-mediated knock-in was reduced but not abolished (as it is without CRISPRs) in the Pprad51-1-2 double mutant, reaching about 30% of the wild type level.…”