A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity

Uringa, Evert-Jan; Baldeyron, Céline; Odijk, Hanny; Wassenaar, Evelyne; Cappellen, Wiggert A. van; Maas, Alex; Hoeijmakers, Jan H.J.; Baarends, Willy M.; Kanaar, Roland; Essers, Jeroen

doi:10.1016/j.dnarep.2014.11.002

Cited by 6 publications

(3 citation statements)

References 66 publications

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“…The Rad51 GFP/WT and Rad54 GFP/− ES cell lines were engineered by gene targeting in E14 ES cells using the knock-in constructs designed to express the fusion protein from endogenous promoter by inserting into exon III ( Rad51 ) or IV ( Rad54 ) of the cDNA encoding the remainder of the coding sequence fused to EGFP followed by a PGK-neo selection cassette. This has been described previously ( Agarwal et al, 2011 ; Uringa et al, 2014 ). Plasmids for the expression of the BRCA2 BRC3 repeat (amino acids 1,415–1,483 of human BRCA2) and a control peptide (BRC3-ΔFK, containing a deletion corresponding to Phe 1428 –Lys 1434 within BRC3), both fused to a nuclear localization signal in pCAGGS expression vector under the CAG promoter, were obtained from J. Stark (Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY) and have been described previously ( Stark et al, 2002 ).…”

Section: Methodsmentioning

confidence: 99%

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Reuter

Zelensky

Smal

et al. 2014

Journal of Cell Biology

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Section: Methodsmentioning

confidence: 99%

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Reuter

Zelensky

Smal

et al. 2014

Journal of Cell Biology

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“…RAD51-GFP in Arabidopsis, mammals and yeast [39,41,[70][71][72] and DMC1-GFP in fission yeast [73]).…”

Section: Plos Geneticsmentioning

confidence: 99%

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

et al. 2022

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Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.

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“…Thus, we assessed whether the DSB repair mechanisms are delayed during myogenic differentiation as observed for the BER pathway. As the fusion of GFP to RAD51 affects HR-mediated DSB repair (Uringa et al, 2015), we studied the recruitment of this central HR protein, which promotes the search for homology and strand pairing steps, by immunostaining. We observed no IRIF of RAD51 in myotubes in contrast to myoblasts, and we did not detect RAD51 in myotubes by immunostaining, as previously reported by RT-qPCR and Western blot in Vahidi Ferdousi et al (2014).…”

Section: Discussionmentioning

confidence: 99%

Decline of DNA damage response along with myogenic differentiation

Sutcu,

Rassinoux,

Donnio

et al. 2023

Preprint

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DNA integrity is incessantly confronted to endogenous and exogenous agents inducing DNA lesions, which are harmful for cellular homeostasis. Luckily all organisms are equipped with a network of DNA damage response (DDR) mechanisms that will repair DNA lesions and restore the proper cellular activities. Despite DNA repair mechanisms have been revealed in vitro and in replicating cells, still little is known on how DNA lesions are repaired and consequently how cellular homeostasis is maintained in post-mitotic cells. Muscle fibers are highly specialised post-mitotic cells organized in syncytia and, they are vulnerable to age-related degeneration and atrophy following radiotherapy treatment. We have here studied in detail the DNA repair capacity of muscle fibers nuclei and compared it with the one measured in proliferative myoblasts. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair (BER), the non-homologous end joining (NHEJ) and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, all of these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For BER and HR, this decline can be linked to reduced steady state levels of key proteins involved in these processes, probably since nuclei within muscle fibers no longer replicate their DNA.

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A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity

Cited by 6 publications

References 66 publications

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Decline of DNA damage response along with myogenic differentiation

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