Rad51 paralog complex Rad55–Rad57 acts as a molecular chaperone during homologous recombination
4
Citation
55
Reference
10
Related Paper
Citation Trend
Abstract:
Summary Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.Keywords:
Chaperone (clinical)
FLP-FRT recombination
The analysis of mutant organisms and cell lines is important in determining the function of specific proteins. Recent technological advances in gene targeting by homologous recombination in mammalian systems enable the production of mutants in any desired gene, and can be used to produce mutant mouse strains and mutant cell lines. The yeast Flp/FRT recombinase system and bacteriophage recombinases such as Cre and its recognition sequence, loxP, allow spatial and temporal control of knockouts. This unit discusses crucial issues for homologous recombination experiments, including requirements for the source of DNA, criteria for the targeting constructs, methods of enrichment for homologous recombinants, (positive and negative selection, and the use of endogenous promoters), and the types of mutations that can be created.
Cre recombinase
Gene targeting
FLP-FRT recombination
Gene knockout
Cre-Lox recombination
Cite
Citations (12)
The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.
FLP-FRT recombination
Non-homologous end joining
Ectopic recombination
Site-specific recombination
Cite
Citations (54)
FLP-FRT recombination
Chinese hamster
Non-homologous end joining
Cite
Citations (70)
The process of homologous recombination promotes error-free repair of double-strand breaks and is essential for meiosis. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Herein, we review recent genetic, biochemical, and structural analyses of the genes and proteins involved in recombination.
RAD52
Non-homologous end joining
FLP-FRT recombination
Ectopic recombination
Rad50
Non-allelic homologous recombination
Mitotic crossover
Cite
Citations (790)
Abstract Background Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants. Results We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility. Conclusion Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.
Gene targeting
FLP-FRT recombination
Heterologous
Cre recombinase
Homology directed repair
Non-homologous end joining
Cite
Citations (12)
The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.
Non-homologous end joining
FLP-FRT recombination
Non-allelic homologous recombination
Homology directed repair
Cite
Citations (25)
Integrases
FLP-FRT recombination
Site-specific recombination
Cre-Lox recombination
Cre recombinase
Cite
Citations (99)
Summary Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.
Chaperone (clinical)
FLP-FRT recombination
Cite
Citations (4)