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del Val, Elsa, William Nasser, Hafid Abaibou und Sylvie Reverchon. „RecA and DNA recombination: a review of molecular mechanisms“. Biochemical Society Transactions 47, Nr. 5 (18.10.2019): 1511–31. http://dx.doi.org/10.1042/bst20190558.
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Abstract Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer–monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.
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Luisi-DeLuca, C., S. T. Lovett und R. D. Kolodner. „Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants.“ Genetics 122, Nr. 2 (01.06.1989): 269–78. http://dx.doi.org/10.1093/genetics/122.2.269.
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Abstract The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.
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Chittela, Rajani Kant, und Jayashree K. Sainis. „Plant DNA Recombinases: A Long Way to Go“. Journal of Nucleic Acids 2010 (2010): 1–10. http://dx.doi.org/10.4061/2010/646109.
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DNA homologous recombination is fundamental process by which two homologous DNA molecules exchange the genetic information for the generation of genetic diversity and maintain the genomic integrity. DNA recombinases, a special group of proteins bind to single stranded DNA (ssDNA) nonspecifically and search the double stranded DNA (dsDNA) molecule for a stretch of DNA that is homologous with the bound ssDNA. Recombinase A (RecA) has been well characterized at genetic, biochemical, as well as structural level from prokaryotes. Two homologues of RecA called Rad51 and Dmc1 have been detected in yeast and higher eukaryotes and are known to mediate the homologous recombination in eukaryotes. The biochemistry and mechanism of action of recombinase is important in understanding the process of homologous recombination. Even though considerable progress has been made in yeast and human recombinases, understanding of the plant recombination and recombinases is at nascent stage. Since crop plants are subjected to different breeding techniques, it is important to know the homologous recombination process. This paper focuses on the properties of eukaryotes recombinases and recent developments in the field of plant recombinases Dmc1 and Rad51.
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Haldenby, Sam, Malcolm F. White und Thorsten Allers. „RecA family proteins in archaea: RadA and its cousins“. Biochemical Society Transactions 37, Nr. 1 (20.01.2009): 102–7. http://dx.doi.org/10.1042/bst0370102.
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Recombinases of the RecA family are essential for homologous recombination and underpin genome stability, by promoting the repair of double-stranded DNA breaks and the rescue of collapsed DNA replication forks. Until now, our understanding of homologous recombination has relied on studies of bacterial and eukaryotic model organisms. Archaea provide new opportunities to study how recombination operates in a lineage distinct from bacteria and eukaryotes. In the present paper, we focus on RadA, the archaeal RecA family recombinase, and its homologues in archaea and other domains. On the basis of phylogenetic analysis, we propose that a family of archaeal proteins with a single RecA domain, which are currently annotated as KaiC, be renamed aRadC.
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García-Vázquez, Francisco A., Salvador Ruiz, Carmen Matás, M. José Izquierdo-Rico, Luis A. Grullón, Aitor De Ondiz, Luis Vieira, Karen Avilés-López, Alfonso Gutiérrez-Adán und Joaquín Gadea. „Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA“. REPRODUCTION 140, Nr. 2 (August 2010): 259–72. http://dx.doi.org/10.1530/rep-10-0129.
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Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.
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Hofstatter, Paulo G., Alexander K. Tice, Seungho Kang, Matthew W. Brown und Daniel J. G. Lahr. „Evolution of bacterial recombinase A ( recA ) in eukaryotes explained by addition of genomic data of key microbial lineages“. Proceedings of the Royal Society B: Biological Sciences 283, Nr. 1840 (12.10.2016): 20161453. http://dx.doi.org/10.1098/rspb.2016.1453.
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Recombinase enzymes promote DNA repair by homologous recombination. The genes that encode them are ancestral to life, occurring in all known dominions: viruses, Eubacteria, Archaea and Eukaryota. Bacterial recombinases are also present in viruses and eukaryotic groups (supergroups), presumably via ancestral events of lateral gene transfer. The eukaryotic recA genes have two distinct origins (mitochondrial and plastidial), whose acquisition by eukaryotes was possible via primary (bacteria–eukaryote) and/or secondary (eukaryote–eukaryote) endosymbiotic gene transfers (EGTs). Here we present a comprehensive phylogenetic analysis of the recA genealogy, with substantially increased taxonomic sampling in the bacteria, viruses, eukaryotes and a special focus on the key eukaryotic supergroup Amoebozoa, earlier represented only by Dictyostelium . We demonstrate that several major eukaryotic lineages have lost the bacterial recombinases (including Opisthokonta and Excavata), whereas others have retained them (Amoebozoa, Archaeplastida and the SAR-supergroups). When absent, the bacterial recA homologues may have been lost entirely (secondary loss of canonical mitochondria) or replaced by other eukaryotic recombinases. RecA proteins have a transit peptide for organellar import, where they act. The reconstruction of the RecA phylogeny with its EGT events presented here retells the intertwined evolutionary history of eukaryotes and bacteria, while further illuminating the events of endosymbiosis in eukaryotes by expanding the collection of widespread genes that provide insight to this deep history.
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Liu, Yu-Tien, Chia-Geun Chen, Der-Chiang Chao, Fan Lee, Ching-Len Liao, Huey-Kang Sytwu, Chi-Fu Chou und Dar-Der Ji. „Sequence analysis of theGluconobacter oxydansRecA protein and construction of arecA-deficient mutant“. Canadian Journal of Microbiology 45, Nr. 4 (01.04.1999): 347–51. http://dx.doi.org/10.1139/w99-009.
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The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, andPseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.Key words: Gluconobacter orphans, recA gene, DNA repair, recA mutant, SOS box.
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Inagaki, Satoko, Kazuyo Fujita, Yukiko Takashima, Kayoko Nagayama, Arifah C. Ardin, Yuki Matsumi und Michiyo Matsumoto-Nakano. „Regulation of Recombination betweengtfB/gtfCGenes inStreptococcus mutansby Recombinase A“. Scientific World Journal 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/405075.
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Streptococcus mutansproduces 3 types of glucosyltransferases (GTFs), whose cooperative action is essential for cellular adhesion. The recombinase A (RecA) protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions.S. mutansstrain MT8148 was grown in the presence of recombinant RecA (rRecA) protein, after which smooth colonies were isolated. The biological functions and sequences of thegtfBandgtfCgenes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of thegtfBandgtfCgenes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of therecAgene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions ofgtfBandgtfCgenes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.
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Pan, Yue, Ningkang Xie, Xin Zhang, Shuo Yang und Shaowu Lv. „Computational Insights into the Dynamic Structural Features and Binding Characteristics of Recombinase UvsX Compared with RecA“. Molecules 28, Nr. 8 (11.04.2023): 3363. http://dx.doi.org/10.3390/molecules28083363.
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RecA family recombinases are the core enzymes in the process of homologous recombination, and their normal operation ensures the stability of the genome and the healthy development of organisms. The UvsX protein from bacteriophage T4 is a member of the RecA family recombinases and plays a central role in T4 phage DNA repair and replication, which provides an important model for the biochemistry and genetics of DNA metabolism. UvsX shares a high degree of structural similarity and function with RecA, which is the most deeply studied member of the RecA family. However, the detailed molecular mechanism of UvsX has not been resolved. In this study, a comprehensive all-atom molecular dynamics simulation of the UvsX protein dimer complex was carried out in order to investigate the conformational and binding properties of UvsX in combination with ATP and DNA, and the simulation of RecA was synchronized with the property comparison learning for UvsX. This study confirmed the highly conserved molecular structure characteristics and catalytic centers of RecA and UvsX, and also discovered differences in regional conformation, volatility and the ability to bind DNA between the two proteins at different temperatures, which would be helpful for the subsequent understanding and application of related recombinases.
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Ramos, Cristina, Rogelio Hernández-Tamayo, María López-Sanz, Begoña Carrasco, Ester Serrano, Juan C. Alonso, Peter L. Graumann und Silvia Ayora. „The RecD2 helicase balances RecA activities“. Nucleic Acids Research 50, Nr. 6 (02.03.2022): 3432–44. http://dx.doi.org/10.1093/nar/gkac131.
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Abstract DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA–ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.
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Kuan, C. T., S. K. Liu und I. Tessman. „Excision and transposition of Tn5 as an SOS activity in Escherichia coli.“ Genetics 128, Nr. 1 (01.05.1991): 45–57. http://dx.doi.org/10.1093/genetics/128.1.45.
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Abstract Excision and transposition of the Tn5 element in Escherichia coli ordinarily appear to occur by recA-independent mechanisms. However, recA(Prtc) genes, which encode RecA proteins that are constitutively activated to the protease state, greatly enhanced excision and transposition; both events appeared to occur concomitantly and without destruction of the donor DNA. The recombinase function of the RecA protein was not required. Transposition was accompanied by partial, and occasionally full, restoration of the functional integrity of the gene vacated by the excised Tn5. The stimulation of transposition was inhibited by an uncleavable LexA protein and was strongly enhanced by an additional role of the RecA(Prtc) protein besides its mediation of LexA cleavage. To account for the enhanced transposition, we suggest that (i) there may be a LexA binding site within the promoter for the IS50 transposase, (ii) activated RecA may cleave the IS50 transposition inhibitor, and (iii) the transposase may be formed by RecA cleavage of a precursor molecule.
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Bonde, Nina J., Zachary J. Romero, Sindhu Chitteni-Pattu und Michael M. Cox. „RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange“. Nucleic Acids Research 50, Nr. 4 (12.02.2022): 2201–10. http://dx.doi.org/10.1093/nar/gkac041.
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Abstract In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.
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Gadea, J., A. Gutierrez-Adan und F. A. Garcia-Vazquez. „303 EFFECT OF THE PRESENCE OF EXOGENOUS DNA AND RECOMBINASE-A PROTEIN ON THE BOAR SPERM FUNCTIONALITY“. Reproduction, Fertility and Development 21, Nr. 1 (2009): 248. http://dx.doi.org/10.1071/rdv21n1ab303.
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Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer based on the intrinsic ability of sperm cells to bind exogenous DNA molecules and to transfer them into the oocyte. It has been reported that coating exogenous DNA with recombinase-A protein (RecA) may improve the efficiency of transgenic embryo production (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). The objective of this study was to investigate the effect of the presence of DNA and DNA-RecA complex on the sperm functionality. Spermatozoa were incubated with linealized plasmid 5.7 kb enhanced green fluorescent protein (DNA) or RecA-DNA complexes (40:1 or 40:3 protein:DNA w/w) in a relation of 108 cells mL–1 and 5 to 15 μg of DNA mL–1. Sperm viability, membrane lipid disorder, reactive oxygen species (ROS) generation, and motion parameters were evaluated by flow cytometry and computer-assisted semen analysis (CASA) for control (no DNA), DNA (5 μg), RecA 5 (5 μg), and RecA 15 (15 μg). A total of 5 replicates were used. Data were analyzed by ANOVA. The sperm viability measured by propidium iodide staining was significantly reduced by the presence of RecA DNA complexes (viable cells 77.5a v. 69.4ab v. 55.6b v. 35.8c%; P < 0.01). Lipid membrane disorder (measured by merocyanine 540 and Yopro 1 staining) were significantly reduced in the group with high exogenous DNA concentration (RecA 15 group), with a reduction of percentage of viable cells and low lipid disorder (67.4a v. 60.0a v. 51.5a v. 24.3b; P < 0.01). However, the ROS generation (measured by 2′,5′-dichlorofluorescein diacetate) was not different in the different experimental groups (fluorescent units 20.3 v. 25.5 v. 26.2 v. 13.9; P = 0.36). The motion parameters measured by CASA were affected by the presence of DNA-RecA complexes with a significant reduction in percentage of motile and progressive motile cells in the group RecA 15 (% progressive motility 46.7a v. 43.9a v. 46.8a v. 20.3b; P < 0.01). Also, it was detected a change in the pattern of motility with an increase in the sperm velocities (velocity average path, velocity straight line, and VCL) in groups DNA and RecA 5 and a reduction of these values when the concentration of DNA was 15 μg mL–1 (VCL μm s–1 97.5a v. 106.4b v. 109.1b v. 58.1c; P < 0.01). The motions parameters (LIN, STR, WOB, ALH, BCF) showed significant lower values for the group RecA 15. These results shown that the presence of DNA and RecA-DNA complex modified the pattern of motility and reduced the viability and functionality of the boar sperm in a concentration dose way. It is necessary to select an adequate DNA and RecA concentration that would protect the sperm functionality and optimize the binding process. Supported by BIOCARM 10BIO2005/01-6463 and AGL2006-03495.
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Huang, Tzu-Wen, und Carton W. Chen. „A recA Null Mutation May Be Generated in Streptomyces coelicolor“. Journal of Bacteriology 188, Nr. 19 (01.10.2006): 6771–79. http://dx.doi.org/10.1128/jb.00951-06.
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ABSTRACT The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.
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Zahradka, Ksenija, Jelena Repar, Damir Đermić und Davor Zahradka. „Chromosome Segregation and Cell Division Defects in Escherichia coli Recombination Mutants Exposed to Different DNA-Damaging Treatments“. Microorganisms 11, Nr. 3 (09.03.2023): 701. http://dx.doi.org/10.3390/microorganisms11030701.
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Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.
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Lee, Andrew J., Masayuki Endo, Jamie K. Hobbs, A. Giles Davies und Christoph Wälti. „Micro-homology intermediates: RecA’s transient sampling revealed at the single molecule level“. Nucleic Acids Research 49, Nr. 3 (21.01.2021): 1426–35. http://dx.doi.org/10.1093/nar/gkaa1258.
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Abstract Recombinase A (RecA) is central to homologous recombination. However, despite significant advances, the mechanism with which RecA is able to orchestrate a search for homology remains elusive. DNA nanostructure-augmented high-speed AFM offers the spatial and temporal resolutions required to study the RecA recombination mechanism directly and at the single molecule level. We present the direct in situ observation of RecA-orchestrated alignment of homologous DNA strands to form a stable recombination product within a supporting DNA nanostructure. We show the existence of subtle and short-lived states in the interaction landscape, which suggests that RecA transiently samples micro-homology at the single RecA monomer-level throughout the search for sequence alignment. These transient interactions form the early steps in the search for sequence homology, prior to the formation of stable pairings at >8 nucleotide seeds. The removal of sequence micro-homology results in the loss of the associated transient sampling at that location.
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Azpiroz, María F., und Magela Laviña. „Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid inEscherichia coliK12“. PeerJ 5 (09.05.2017): e3293. http://dx.doi.org/10.7717/peerj.3293.
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RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in theEscherichia coliK12 background. The first model was a smallE. coligenomic island which had been shown to be mobile in its strain of origin and, when cloned, also in theE. coliK12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision inE. coliK12 mutants affected in a number of recombination functions, including the 16E. coliK12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding fromSaccharomyces cerevisiae,which flanked thecatgene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events inE. coliK12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.
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Cao, Y., und T. Kogoma. „The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.“ Genetics 139, Nr. 4 (01.04.1995): 1483–94. http://dx.doi.org/10.1093/genetics/139.4.1483.
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Abstract The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.
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Cai, Yuan, Tianlin Cheng, Yichuan Yao, Xiao Li, Yuqian Ma, Lingyun Li, Huan Zhao et al. „In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway“. Science Advances 5, Nr. 4 (April 2019): eaav3335. http://dx.doi.org/10.1126/sciadv.aav3335.
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Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)–based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.
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Meyers, Paul R. „Analysis of recombinase A (recA/RecA) in the actinobacterial family Streptosporangiaceae and identification of molecular signatures“. Systematic and Applied Microbiology 38, Nr. 8 (Dezember 2015): 567–77. http://dx.doi.org/10.1016/j.syapm.2015.10.001.
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Stohl, Elizabeth A., Joel P. Brockman, Kristin L. Burkle, Katsumi Morimatsu, Stephen C. Kowalczykowski und H. Steven Seifert. „Escherichia coliRecX Inhibits RecA Recombinase and Coprotease Activitiesin Vitroandin Vivo“. Journal of Biological Chemistry 278, Nr. 4 (09.11.2002): 2278–85. http://dx.doi.org/10.1074/jbc.m210496200.
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Long, Eric O., und Malcolm J. W. Sim. „The Human NK Cell Receptor KIR2DS4 Detects a Conserved Bacterial Epitope Presented by HLA-C“. Journal of Immunology 202, Nr. 1_Supplement (01.05.2019): 177.24. http://dx.doi.org/10.4049/jimmunol.202.supp.177.24.
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Abstract Natural killer (NK) cells are innate immune effector cells regulated by many germline-encoded activating and inhibitory receptors. The DAP12-associated activating receptor KIR2DS4 has been linked with multiple disease processes including cancer, disorders of pregnancy, and resistance to HIV. However, the precise ligands for KIR2DS4 remain poorly defined and the role of this receptor in immune responses is unclear. Here we show that human KIR2DS4 is a highly peptide-specific receptor for the human MHC-I molecule HLA-C*05:01. Of over 60 different peptides tested, only two conferred strong binding to KIR2DS4. Recognition of these peptides presented by HLA-C*05:01 potently activated KIR2DS4+ NK cells to degranulate and produce IFN-gamma and TNF-alpha. A recombinant peptide:HLA-C complex was sufficient to activate KIR2DS4+ NK cells. An alignment search of the KIR2DS4 binding peptides identified an epitope in recombinase A (RecA), a highly conserved bacterial protein. RecA epitopes from Chlamydia, Campylobacter, Brucella and Helicobacter pathogens were presented by HLA-C* 05:01, bound KIR2DS4 and activated KIR2DS4+ NK cells. By sequence alignment we predict that hundreds of species of bacteria contain RecA epitopes that can be presented by HLA-C*05:01 and bind KIR2DS4. These data provide clear evidence that KIR2DS4 is a highly peptide specific activating receptor and suggest that KIR2DS4 evolved to play a role in immune defense to bacteria.
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Paul, Tapas, Andrew F. Voter, Rachel R. Cueny, Momčilo Gavrilov, Taekjip Ha, James L. Keck und Sua Myong. „E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands“. Nucleic Acids Research 48, Nr. 12 (25.05.2020): 6640–53. http://dx.doi.org/10.1093/nar/gkaa442.
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Abstract G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.
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Sauvageau, Synthia, Alicja Z. Stasiak, Isabelle Banville, Mickaël Ploquin, Andrzej Stasiak und Jean-Yves Masson. „Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments“. Molecular and Cellular Biology 25, Nr. 11 (01.06.2005): 4377–87. http://dx.doi.org/10.1128/mcb.25.11.4377-4387.2005.
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ABSTRACT Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.
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Garzón, A., D. A. Cano und J. Casadesús. „Role of Erf recombinase in P22-mediated plasmid transduction.“ Genetics 140, Nr. 2 (01.06.1995): 427–34. http://dx.doi.org/10.1093/genetics/140.2.427.
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Abstract In the absence of host RecA function, plasmid transduction by bacteriophage P22 can be mediated by Erf recombinase. Erf is not carried on the infecting particle but synthesized upon infection. In the recipient cell, Erf can promote both generalized plasmid transduction (which requires the circularization of plasmids transduced as linear multimers) and specialized plasmid transduction (which requires the release of plasmid DNA from linear plasmid-phage cointegrates). Both processes of Erf-mediated plasmid transduction require host RecBCD function. In contrast, RecBCD is not required for Erf-mediated circularization of P22 DNA.
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Baitin, Dmitry M., Irina V. Bakhlanova, Yury V. Kil, Michael M. Cox und Vladislav A. Lanzov. „Distinguishing Characteristics of Hyperrecombinogenic RecA Protein from Pseudomonas aeruginosa Acting in Escherichia coli“. Journal of Bacteriology 188, Nr. 16 (15.08.2006): 5812–20. http://dx.doi.org/10.1128/jb.00358-06.
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ABSTRACT In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecAPa) exhibits a more robust recombinase activity than its E. coli counterpart (RecAEc). Low-level expression of RecAPa in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecAEc. This genetic effect is supported by the biochemical finding that the RecAPa protein is more efficient in filament formation than RecA K72R, a mutant protein with RecAEc-like DNA-binding ability. Expression of RecAPa also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecAPa filaments initiate recombination equally from both the 5′ and 3′ ends. Besides, these filaments exhibit more resistance to disassembly from the 5′ ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.
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Ilatovsky, Andrey V., und Vladislav A. Lanzov. „DNA Repeats in Bacterial Genome and Intracellular Activity of Homologous Recombinase“. Ecological genetics 9, Nr. 1 (15.03.2011): 62–69. http://dx.doi.org/10.17816/ecogen9162-69.
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In present work distribution of perfect direct and inverted repeats in a set of bacterial genomes was analysed. Complementary cumulative distribution functions of repeat frequency for 36 bacterial strains are nonrandom and have certain characteristic features. Inverse relation of direct repeats frequency to recombinogenic activity is shown for reference E. сoli К-12 strain and P. aeruginosa strain with hyperrecombinogenic RecA protein. In general, direct repeat frequency is higher in nonpathogenic strains than that in pathogens.
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Boyer, Benjamin, Claudia Danilowicz, Mara Prentiss und Chantal Prévost. „Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination“. Nucleic Acids Research 47, Nr. 15 (02.08.2019): 7798–808. http://dx.doi.org/10.1093/nar/gkz667.
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Abstract Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.
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Kang, J. H., J. Y. Won und H. Shim. „331 MODIFIED SINGLE-STRANDED OLIGONUCLEOTIDE-RECOMBINASE COMPLEX MEDIATES GENE TARGETING IN MOUSE EMBRYOS“. Reproduction, Fertility and Development 17, Nr. 2 (2005): 316. http://dx.doi.org/10.1071/rdv17n2ab331.
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Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.
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Palmieri, Claudio, Marina Mingoia, Orietta Massidda, Eleonora Giovanetti und Pietro E. Varaldo. „Streptococcus pneumoniae Transposon Tn1545/Tn6003Changes to Tn6002Due to Spontaneous Excision in Circular Form of theerm(B)- andaphA3-Containing Macrolide-Aminoglycoside-Streptothricin (MAS) Element“. Antimicrobial Agents and Chemotherapy 56, Nr. 11 (13.08.2012): 5994–97. http://dx.doi.org/10.1128/aac.01487-12.
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ABSTRACTThe macrolide-aminoglycoside-streptothricin (MAS) element, an ∼4.2-kb insertion containingerm(B) andaphA3resistance determinants, distinguishesStreptococcus pneumoniaetransposon Tn1545/Tn6003from Tn6002. Here, it is shown to be an unstable genetic element that, although it lacks recombinase genes, can exploit long,erm(B)-containing direct repeats acting asattsites for spontaneous excision that may result in loss. Consequent to excision, which is RecA independent, Tn1545/Tn6003changes to Tn6002. In pneumococcal populations harboring Tn1545/Tn6003, the latter appears to coexist with Tn6002.
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Cazaux, Christophe, Jean-Sébastien Blanchet, Delphine Dupuis, Giuseppe Villani, Martine Defais und Neil P. Johnson. „Investigation of the Secondary DNA-binding Site of the Bacterial Recombinase RecA“. Journal of Biological Chemistry 273, Nr. 44 (30.10.1998): 28799–804. http://dx.doi.org/10.1074/jbc.273.44.28799.
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Schons-Fonseca, Luciane, Milena D. Lazova, Janet L. Smith, Mary E. Anderson und Alan D. Grossman. „Beneficial and detrimental genes in the cellular response to replication arrest“. PLOS Genetics 18, Nr. 12 (27.12.2022): e1010564. http://dx.doi.org/10.1371/journal.pgen.1010564.
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DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called “roadblocks” (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.
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Bringel, Françoise, Anna Castioni, Daniel K. Olukoya, Giovanna E. Felis, Sandra Torriani und Franco Dellaglio. „Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices“. International Journal of Systematic and Evolutionary Microbiology 55, Nr. 4 (01.07.2005): 1629–34. http://dx.doi.org/10.1099/ijs.0.63333-0.
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Fourteen strains isolated from vegetable sources and identified as belonging to Lactobacillus plantarum presented an atypical pattern of amplification with a species-specific multiplex-PCR assay. Phylogenetic analysis of two protein-encoding genes, recA (encoding the recombinase A protein) and cpn60 (encoding the GroEL chaperonin), as well as phenotypic and genomic traits revealed a homogeneous group of very closely related strains for which subspecies status is proposed, with the name Lactobacillus plantarum subsp. argentoratensis. The type strain is DKO 22T (=CIP 108320T=DSM 16365T).
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Candelli, Andrea, Mauro Modesti, Erwin J. G. Peterman und Gijs J. L. Wuite. „Single-molecule views on homologous recombination“. Quarterly Reviews of Biophysics 46, Nr. 4 (09.09.2013): 323–48. http://dx.doi.org/10.1017/s0033583513000073.
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AbstractAll organisms need homologous recombination (HR) to repair DNA double-strand breaks. Defects in recombination are linked to genetic instability and to elevated risks in developing cancers. The central catalyst of HR is a nucleoprotein filament, consisting of recombinase proteins (human RAD51 or bacterial RecA) bound around single-stranded DNA. Over the last two decades, single-molecule techniques have provided substantial new insights into the dynamics of homologous recombination. Here, we survey important recent developments in this field of research and provide an outlook on future developments.
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Tessman, E. S., und P. K. Peterson. „Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12.“ Journal of Bacteriology 163, Nr. 2 (1985): 688–95. http://dx.doi.org/10.1128/jb.163.2.688-695.1985.
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Meah, Y. S., und F. R. Bryant. „Activation of a recombinase-deficient mutant recA protein with alternate nucleoside triphosphate cofactors.“ Journal of Biological Chemistry 268, Nr. 32 (November 1993): 23991–96. http://dx.doi.org/10.1016/s0021-9258(20)80483-9.
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SEKTAS, MARIAN, MAGDALENA GREGOROWICZ, MAGDALENA KUCHARSKA und EWA JODELKO. „Integrative Vectors for Gene Deletion and Replacement“. Polish Journal of Microbiology 62, Nr. 1 (2013): 77–80. http://dx.doi.org/10.33073/pjm-2013-010.
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An improved method for gene deletion or replacement in Escherichia coli was developed. It employs a set of integrative vectors and two helper plasmids, as a temporary source of RecA and Flp activities. The integrative vectors combine several useful features including three different selection markers placed between two parallel oriented Flp recombinase target (FRT) sites. Each marker is flanked by two MCSs, for cloning the chosen homologous fragments of DNA to gene targeting. The vectors contain two properly oriented E. coli Chi sites for recombination enhancement. When required, selection markers can be excised from the chromosome resulting in unmarked strains.
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Sciochetti, Stephen A., Patrick J. Piggot, David J. Sherratt und Garry Blakely. „The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning“. Journal of Bacteriology 181, Nr. 19 (01.10.1999): 6053–62. http://dx.doi.org/10.1128/jb.181.19.6053-6062.1999.
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ABSTRACT The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers inB. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripXstrains by the first division after the initiation of germination. The introduction of a recA mutation into ripXstrains resulted in only slight modifications of the ripXphenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing adif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.
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Handa, Naofumi, Asao Ichige, Kohji Kusano und Ichizo Kobayashi. „Cellular Responses to Postsegregational Killing by Restriction-Modification Genes“. Journal of Bacteriology 182, Nr. 8 (15.04.2000): 2218–29. http://dx.doi.org/10.1128/jb.182.8.2218-2229.2000.
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ABSTRACT Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in anEscherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC,recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted “nonself” DNA and repair restricted “self” DNA.
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Defais, Martine, Emilie Phez und Neil P. Johnson. „Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA“. Journal of Biological Chemistry 278, Nr. 6 (26.11.2002): 3545–51. http://dx.doi.org/10.1074/jbc.m204341200.
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Du, Liqin, und Yu Luo. „Structure of a filament of stacked octamers of human DMC1 recombinase“. Acta Crystallographica Section F Structural Biology and Crystallization Communications 69, Nr. 4 (28.03.2013): 382–86. http://dx.doi.org/10.1107/s1744309113005678.
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Eukaryal DMC1 proteins play a central role in homologous recombination in meiosis by assembling at the sites of programmed DNA double-strand breaks and carrying out a search for allelic DNA sequences located on homologous chromatids. They are close homologs of eukaryal Rad51 and archaeal RadA proteins and are remote homologs of bacterial RecA proteins. These recombinases (also called DNA strand-exchange proteins) promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. An octameric form of a truncated human DMC1 devoid of its small N-terminal domain (residues 1–83) has been crystallized. The structure of the truncated DMC1 octamer is similar to that of the previously reported full-length DMC1 octamer, which has disordered N-terminal domains. In each protomer, only the ATP cap regions (Asp317–Glu323) show a noticeable conformational difference. The truncated DMC1 octamers further stack with alternate polarity into a filament. Similar filamentous assemblies of DMC1 have been observed to form on DNA by electron microscopy.
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Wang, Xulin, Zhengqing Xie, Zhaoran Tian, Shuaipeng Wang, Gongyao Shi, Weiwei Chen, Gangqiang Cao et al. „BrDMC1, a Recombinase Gene, Is Involved in Seed Germination in Brassica rapa under Salt Stress“. Agronomy 13, Nr. 2 (18.02.2023): 595. http://dx.doi.org/10.3390/agronomy13020595.
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Recombinases are in part responsible for homologous recombination and genome integrity during DNA repair. DMC1 has a typical RecA domain, and belongs to the recombinase superfamily. The reactive oxygen species (ROS) as a potent DNA damage agent is produced during seed germination under stress conditions. DNA repair should be initiated immediately to allow for subsequent seedling development. In this study, we attempted to characterize the underlying mechanism of BrDMC1 responsiveness to salinity stress using the RNA interference approach in Brassica rapa (B. rapa). Bioinformatics and expression pattern analysis revealed that BrDMC1 only retained BrDMC1.A01 after the whole genome triplication (WGT) event and was primarily transcribed in flowers and seeds. BrDMC1 had high activity in the promoter region during germination, according to histochemical GUS staining. The data showed that salt treatment reduced the germination rate, weakened seed vigor and decreased antioxidant enzyme activity, but increased oxidative damage in BrDMC1-RNAi seeds. Furthermore, the expression of stress-responsive genes and damage repair genes was significantly different in transgenic lines exposed to salt stress. Therefore, BrDMC1 may respond to salt stress by controlling seed germination and the expression of stress-related and damage repair genes in B. rapa.
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Sim, Malcolm J. W., Sumati Rajagopalan, Daniel M. Altmann, Rosemary J. Boyton, Peter D. Sun und Eric O. Long. „Human NK cell receptor KIR2DS4 detects a conserved bacterial epitope presented by HLA-C“. Proceedings of the National Academy of Sciences 116, Nr. 26 (28.05.2019): 12964–73. http://dx.doi.org/10.1073/pnas.1903781116.
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Natural killer (NK) cells have an important role in immune defense against viruses and cancer. Activation of human NK cell cytotoxicity toward infected or tumor cells is regulated by killer cell immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen class I (HLA-I). Combinations of KIR with HLA-I are genetically associated with susceptibility to disease. KIR2DS4, an activating member of the KIR family with poorly defined ligands, is a receptor of unknown function. Here, we show that KIR2DS4 has a strong preference for rare peptides carrying a Trp at position 8 (p8) of 9-mer peptides bound to HLA-C*05:01. The complex of a peptide bound to HLA-C*05:01 with a Trp at p8 was sufficient for activation of primary KIR2DS4+NK cells, independent of activation by other receptors and of prior NK cell licensing. HLA-C*05:01+cells that expressed the peptide epitope triggered KIR2DS4+NK cell degranulation. We show an inverse correlation of the worldwide allele frequency of functionalKIR2DS4with that ofHLA-C*05:01, indicative of functional interaction and balancing selection. We found a highly conserved peptide sequence motif for HLA-C*05:01–restricted activation of human KIR2DS4+NK cells in bacterial recombinase A (RecA). KIR2DS4+NK cells were stimulated by RecA epitopes from multiple human pathogens, includingHelicobacter,Chlamydia,Brucella, andCampylobacter.We predict that over 1,000 bacterial species could activate NK cells through KIR2DS4, and propose that human NK cells also contribute to immune defense against bacteria through recognition of a conserved RecA epitope presented by HLA-C*05:01.
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Shinohara, Miki, Kazuko Sakai, Akira Shinohara und Douglas K. Bishop. „Crossover Interference in Saccharomyces cerevisiae Requires a TID1/RDH54- and DMC1-Dependent Pathway“. Genetics 163, Nr. 4 (01.04.2003): 1273–86. http://dx.doi.org/10.1093/genetics/163.4.1273.
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Abstract Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Δ mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Δ cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.
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Kil, Yuri V., Dmitry M. Baitin, Ryoji Masui, Elizaveta A. Bonch-Osmolovskaya, Seiki Kuramitsu und Vladislav A. Lanzov. „Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus“. Journal of Bacteriology 182, Nr. 1 (01.01.2000): 130–34. http://dx.doi.org/10.1128/jb.182.1.130-134.2000.
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ABSTRACT The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon,Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min−1 at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C).
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Zahradka, Ksenija, Jelena Repar, Damir Đermić und Davor Zahradka. „Genetic analysis of transductional recombination in Escherichia coli reveals differences in the postsynaptic stages of RecBCD and RecFOR pathways“. Periodicum Biologorum 124, Nr. 3-4 (05.05.2023): 97–106. http://dx.doi.org/10.18054/pb.v124i3-4.23604.
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Background and purpose: Homologous recombination in Escherichia coli proceeds via two pathways, RecBCD and RecFOR, which use different enzymes for DNA end resection and loading of RecA recombinase. The postsynaptic reactions following RecA-mediated homologous pairing have mostly been studied within the RecBCD pathway. They involve RuvABC helicase/resolvase complex, RecG and RadA helicases that process recombination intermediates to produce recombinant DNA molecules. Also, RecG functionally interacts with the PriA protein in initiation of recombination-dependent replication. Here, we studied the individual and combined effects of ruvABC, recG and radA null mutations on transductional recombination in both pathways. The effect of the priA300 mutation, which acts as a suppressor of the recG mutation, was also tested. The goal was to characterize the postsynaptic stage of transductional recombination in more details, especially in the RecFOR pathway, which is less well-studied. Materials and methods: Phage P1vir-mediated transduction was used to measure recombination efficiency in a series of recombination mutants. The proA+ marker was used for selection in transductional crosses with various ΔproA recipients. Results: The ruvABC mutation moderately decreased recombination in both recombination pathways, while radA had no effect. The recG mutation reduced recombination in the RecBCD pathway but not in the RecFOR pathway. The strong recombination defect of recG radA double mutants in both pathways was completely suppressed by the priA300 mutation, and this suppression depended on the functional RuvABC complex. Conclusions: RecG and RadA proteins have a redundant role in transductional recombination via RecFOR pathway. In both recombination pathways, RecG and RadA functionally interact with PriA, probably during initiation of recombination-dependent replication.
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Xia, S. J., M. A. Shammas und R. J. Shmookler Reis. „Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase.“ Molecular and Cellular Biology 17, Nr. 12 (Dezember 1997): 7151–58. http://dx.doi.org/10.1128/mcb.17.12.7151.
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Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two- to fivefold. T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.
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Raschle, Markus, Stephen Van Komen, Peter Chi, Tom Ellenberger und Patrick Sung. „Multiple Interactions with the Rad51 Recombinase Govern the Homologous Recombination Function of Rad54“. Journal of Biological Chemistry 279, Nr. 50 (30.09.2004): 51973–80. http://dx.doi.org/10.1074/jbc.m410101200.
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In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Δ129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.
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Bryant, F. R. „Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity.“ Journal of Biological Chemistry 263, Nr. 18 (Juni 1988): 8716–23. http://dx.doi.org/10.1016/s0021-9258(18)68364-4.
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Khoo, Kelvin H. P., Hayley R. Jolly und Jason A. Able. „The RAD51 gene family in bread wheat is highly conserved across eukaryotes, with RAD51A upregulated during early meiosis“. Functional Plant Biology 35, Nr. 12 (2008): 1267. http://dx.doi.org/10.1071/fp08203.
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The RADiation sensitive protein 51 (RAD51) recombinase is a eukaryotic homologue of the bacterial Recombinase A (RecA). It is required for homologous recombination of DNA during meiosis where it plays a role in processes such as homology searching and strand invasion. RAD51 is well conserved in eukaryotes with as many as four paralogues identified in vertebrates and some higher plants. Here we report the isolation and preliminary characterisation of four RAD51 gene family members in hexaploid (bread) wheat (Triticum aestivum L.). RAD51A1, RAD51A2 and RAD51D were located on chromosome group 7, and RAD51C was on chromosome group 2. Q-PCR gene expression profiling revealed that RAD51A1 was upregulated during meiosis with lower expression levels seen in mitotic tissue, and bioinformatics analysis demonstrated the evolutionary linkages of this gene family to other eukaryotic RAD51 sequences. Western blot analysis of heterologously expressed RAD51 from bread wheat has shown that it is detectable using anti-human RAD51 antibodies and that molecular modelling of the same protein revealed structural conservation when compared with yeast, human, Arabidopsis and maize RAD51A orthologues. This report has widened the knowledge base of this important protein family in plants, and highlighted the high level of structural conservation among RAD51 proteins from various species.