Molecular interaction and cellular location of reca and. Phylogenetic trees of all currently available complete reca proteins were inferred using multiple maximum parsimony and distance matrix methods. Study of interactions of dna with reca and other proteins by. Get a printable copy pdf file of the complete article 1. Probing the dna sequence specificity of escherichia coli. The primary function of bacterial recombination systems is the nonmutagenic repair of stalled or collapsed replication forks. The reca protein plays a central role in various steps of homologous recombination, in the repair of broken dna molecules and in the restart of stalled replication forks. Therefore, understanding factors that affect reca structure and stability is broadly applicable to many fields.
Reca protein is the prototypical strandexchange protein of e. Recombinational dna repair in bacteria and the reca protein. Pdf the bacterial reca protein is the prototypical recombinase, promoting the central. Reca is also a target for inhibiting microbial antibiotic resistance, understanding cancer propagation, and characterizing neurodegenerative disorders. Postreplication kevin p rice,university of wisconsinmadison, madison, wisconsin, usa michael m cox,university of wisconsinmadison, madison, wisconsin, usa recombinational dna repair represents the primary function for homologous dna. The reca protein can be loaded onto the ssbpcoated 3 overhang in one of two distinct pathways, one that requires the recfor enzyme or one that requires the recor enzyme. Proteins and other charged biological polymers migrate in an electric field. A reca structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous dna repair proteins. A change in the genes dna sequence may lead to a change in the amino acid sequence of the protein. The mutations are intragenic suppressors of the reca1202 allele and were detected by the reduced coprotease activity of the gene product. Reca or recalike proteins are ubiquitous in nearly all cells and are essential for the processes of homologous recombination, recombinational. It assembles on singlestranded dna to form a helical nucleoprotein. The recor proteins modulate reca protein function at 5.
In bacteria, the major function of homologous genetic recombination is recombinational dna repair. Hatched lines shows the location of gcat transition zones. Reca promotes the autodigestion of the lexa repressor, umud protein and lambda repressor. Abstractthe bacteriophage p1 recombination enhancement function ref protein is a recadependent. Singlemolecule imaging of dna pairing by reca reveals a. Structure, function, and regulation chapter pdf available in topics in current genetics 17. The recf pathway, also called the recfor pathway, is a pathway of homologous recombination that repairs dna in bacteria. Recadependent programmable endonuclease ref cleaves dna in. In the presence of singlestranded dna ssdna, generated, for instance, by direct or indirect dna damage, reca becomes activated reca. Reca protein promotes dna strand exchange, a reaction that contributes to fork regression and dna end invasion steps. The escherichia coli protein reca catalyzes the strand exchange reaction used in dna repair and genetic recombination. The homologous protein is called rad51 in eukaryotes and rada in archaea reca has multiple activities, all related to dna repair.
Homologs of reca protein exist in all classes of organisms, where they play central roles in homologous genetic recombination and recombinational dna repair. In escherichia coli the lexa protein acts as a reca repressor and is able, in response to dna. Its main function which has been most widely studied is that it facilitates dna recombination in order to repair dna double strand breaks. While reca is highly conserved, the mechanism of transcriptional regulation of its structural gene is less conserved. We have determined the nucleotide sequence of the reca gene of escherichia coli. Reca is a highly conserved prokaryotic protein that not only plays several important roles connected to dna metabolism but also affects the cell response to various stress conditions. A ubiquitous protein that is essential for this dna templatedirected repair is reca 1.
Cox department of biochemistry we characterize aspects of the conformation and dynamic state of reca university of. Probing the dna sequence specificity of escherichia coli reca. Reca protein plays a role in the chemotactic response and. Livnehthe mutagenesis protein umuc is a dna polymerase.
The translocating recbcd enzyme stimulates recombination by directing reca protein onto ssdna in a chiregulated manner. Bacterial reca protein promotes adenoviral recombination. The reca removal activity of uvrd is slowed by reca variants. Recauk has a sales force of over 80 sales professionals giving nationwide coverage and offering technical advice on over 5,500 product lines. The extreme thermostability makes tth reca ideal for molecular biology applications that require an elevated temperature condition, such as nucleic acid amplification and sequencing. The effect of two reca alleles on the multiplicity reactivation pathway of mitomycin c damaged bacteriophage t4 was studied. Probing bufferspecific effects on nucleotide binding to. Secondary structure the primary sequence or main chain of the protein must organize itself to form a compact structure. As an independent governing authority, we set, regulate, and enforce standards for real estate brokerage, mortgage brokerage, property management and real estate appraisal professionals across the. The sequence of the reca gene shows that the reca protein is composed of 352 amino acids and has a molecular weight of 37,852 horii et al. Reca protein, which is essential for genetic recombination in escherichia coli, was extensively purified from a strain of e. Even changing just one amino acid in a protein s sequence can affect the protein s overall structure and function.
The protein binds to singlestranded ss dna to form a nucleoprotein filament that exhibits a helical structure. Genotype of the embryos used for injection is shown. The reca protein catalyses the atpdriven homologous pairing and strand exchange of dna molecules. The bacterial reca protein and the recombinational dna. Cys3 asp20 asn15 met9 thr17 ser20 glu30 gln pro10 gly35 ala38 val22 ile27 leu31 tyr7 phe10 his2lys27 trp2. Michel, 2005 acquiring coprotease activity that prompts autocleavage of the lexa repressor which governs. In this reaction, reca first polymerizes on singlestranded dna ssdna to form a righthanded helical filament with one monomer per 3 nt of ssdna.
Dna of the gene that encodes the protein or that encodes a portion of the protein, for multisubunit proteins. Basal level of reca protein in wildtype bacteria and re pairdeficient mutants. Pdf the reca protein is a recombinase functioning in recombinational dna repair in bacteria. In the recfor pathway, the recfr complex binds where the singlestrand dna of the 3 meets the doublestrand dna. Reca protein advanced article university of california. In addition to its role in dna damage repair and recombination, the reca protein, through its interaction with chew, is involved in swarming motility, a form of flagelladependent movement across surfaces. However, much of the coated cssdna failed to migrate into the agarose gel, suggesting the nls reca gal4 filaments form higher order structures, possibly via the dimerization. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a pdf plugin installed and enabled in your browser. Pdf regulation of bacterial reca protein function researchgate. The coding region of the reca gene has 1059 base pairs, which specify 352 amino acids. Reca uk are operating on reduced staff during the covid19 restrictions.
The crystal structures of mycobacterium smegmatis reca reca ms and its complexes with adp, atpgammas, and datp show that reca ms has an expanded binding site like that in mycobacterium tuberculosis reca, although there are small differences between the proteins in their modes of nucleotide binding. It repairs breaks that occur on only one of dnas two strands, known as singlestrand gaps. A reca protein surface required for activation of dna. Reca protein piero r bianco,university of california, davis, california, usa stephen c kowalczykowski,university of california, davis, california, usa the reca protein of escherichia coli is the prototypic dna strand exchange protein. Adenovirus types grouped in the same proteotypes are distinguished by a vertical gap. Structural biochemistryreca protein wikibooks, open. Reca protein advanced article university at buffalo. Structural knowledge of mtreca is imperative for a full understanding of both these activities and any ensuing application. It was shown that both primary functions attributed to the enzyme, that of catalyzing homologous pairing and strand transfer reactions of joint molecule formation and that of a highly specific protease, are required for full function of the pathway. The removal of reca from dna also requires atp hydrolysis by the uvrd helicase but not by reca protein.
It assembles on singlestranded dna to form a helical nucleoprotein filament that is the. There are two hypervariable regions in the penton base gene, two in the hexon gene, and one in the fiber gene. Use of reca fusion proteins to induce genomic modifications. Umud and reca directly modulate the mutagenic potential of. Reca protein plays a role in the chemotactic response. Purification and characterization of the reca protein from neisseria. The optimum ph for binding of reca protein to doublestanded dna is about ph 6. Another key protein involved in regulating the sos response is reca. The recf pathway can also repair doublestrand breaks in dna when the recbcd pathway, another pathway of homologous recombination in bacteria, is inactivated by mutations. In vitro studies indicate that in the presence of atp, reca promotes the strand. Reca protein is known to bind to both singlestranded dna and doublestranded dna 21,2426. The reca protein plays a central role in these repair pathways, and its biochemistry must be considered in this context. Reca protein blue wrapped around two strands of dna orange and pink.
The bacterial reca protein and the recombinational dna repair. It has a ssdnadependent atpase activity at an optimal temperature between 6575c. Frontiers molecular interaction and cellular location of. Reca is a multifunctional protein present in almost all members of the bacteria domain eisen, 1995. This supports the proposal for a passive role of recaloading machineries in ssb removal. Mechanism of homologous recombination from the recassdna. As an independent governing authority, we set, regulate, and enforce standards for real estate brokerage, mortgage brokerage, property management and real estate appraisal professionals across the province. Reca protein is presented as a linear amino acid sequence. In vitro studies towards understanding the function and.
Tth reca is a reca homolog isolated from thermus thermophilus. Reca has multiple functions, all of which are related to dna repair. The recaremoval activity of uvrd is slowed by reca variants. The recbc enzyme, which lacks the recd subunit, loads reca protein constitutively, in a manner that is independent of using a. This article is from nucleic acids research, volume 42. Reca protein piero r bianco, university of california, davis, california, usa stephen c kowalczykowski, university of california, davis, california, usa the reca protein of escherichia coli is the prototypic deoxyribonucleic acid dna strand exchange protein. Twentyeight reca mutants, isolated after spontaneous mutagenesis generated by the combined action of reca1202prtc and umudc proteins, were characterized and sequenced. In vitro studies indicate that in the presence of atp, reca promotes the strand exchange of singlestrand dna fragments with homologous duplex dna. The reca monomers first polymerize to form a helical filament around ssdna. Suramin is a potent and selective inhibitor of mycobacterium. Abstractthe bacteriophage p1 recombination enhancement function ref protein is a reca dependent.
Identification of residues in the l1 region of the reca protein which. In escherichia coli, at least two groups of proteins, or recombination machines, can operate independently on broken dna to produce a 3terminated singlestranded dna filament coated with reca protein and ready for synapsis with intact homologous dna. Pdf the reca protein of escherichia coli is the prototypic deoxyribonucleic acid dna strand exchange protein. These are highlighted in green and red, and the amino acids corresponding to each domain are shown at the top of each domain. Comparison and analysis of the trees reveal that the inferred relationships among these proteins are highly robust. The recorfacilitated binding of reca protein to singlestranded dna ssdna that is bound by singlestranded dnabinding protein ssb is much faster if the. Escherichia coli reca protein catalyzes the central dna strandexchange step of homologous recombination, which is essential for the repair of doublestranded dna breaks. The reca family of atpases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. After resection of broken dna to produce singlestranded dna ssdna, reca assembles on this ssdna into a. The reca protein of escherichia coli is essential for homol ogous genetic recombination and for the postreplicative repair of damaged dna radding, 1982. All of these proteins function in a network that determines where and how reca functions.
The reca protein is a critical enzyme in this process, as it catalyzes the pairing of ssdna with complementary regions of dsdna. Reca protein promotes the central steps ofany recombi national process, including the alignment oftwo homo logous dna molecules and the subsequent exchange of. Reca, a ubiquitous multifunctional protein, is a key component of the processes of homologous genetic recombination and dna repair. The reca group, with over 500 million turnover, offer massive resources to help give you, the customer, the best possible service. Polypeptide sequences can be obtained from nucleic acid sequences. This is done in an elegant fashion by forming secondary structure elements the two most common secondary structure elements are alpha helices and beta sheets, formed by repeating amino acids with the same. Recadependent programmable endonuclease ref cleaves dna. Members of reca family, including the rada protein of archaea and the rad51 and dmc1 proteins of eukaryotes, share a core atpase domain. Alternatively, you can download the file locally and open with any standalone pdf reader.
B injection of complementary ssdnanlsrecagal4 filaments leads to loh at the gol locus, resulting in mosaic eye pigmentation. Colocalization of viral dna and each rec protein is suggested by the yellow color. The broken strand is first processed by other proteins described below to expose a. The small white boxes in the micrographs show the locations of the insets. The evolution of the reca protein was analyzed using molecular phylogenetic techniques. Dorsal views of eyes at 3 dpf showing wildtype pigmentation patterns in a noninjected embryo upper panel and mosaic.
Amino terminal sequence of the reca protein of escherichia. Similar to reca, the nls reca gal4 protein also caused a mobility shift of the cssdna, indicating that the nls reca gal4 fusion protein retained the ability to bind cssdna. Reca monomers polymerize onto dna in the presence of atp to form a nucleoprotein filament that is competent to promote dna strand exchange and induce the sos response. There are 8 highly conserved subdomains found in all reca protein homologues. Genetic recombination in escherichia coli is stimulated by the recombination hotspot chi. The reca protein as a model molecule for molecular.
The escherichia coli reca protein is the prototype of a bacterial recombinase common to every species of freeliving bacteria. Knightmutations at pro67 in the reca protein ploop. The real estate council of alberta regulates real estate industry professionals in alberta. This is not a process reserved only for rare doublestrand breaks caused by. This function makes it possible to complete recombination. The structural parameters for functioning reca nucleoprotein filaments in vitro have been derived largely from electron microscopy studies figure 3. The reca protein can catalyze one directional branch migration. The bacterial reca protein and the recombinational dna repair of stalled replication forks. The pribnow and shinedalgarno sequences well as the codons used for initiation and termination of translation are boxed. Realtime observation of reca filament dynamics with single. Monomers in the crystal structure pack so as to form a righthanded helical filament with six monomers per turn figure 2a. Presentation mode open print download current view.
Interaction of reca protein with pbr322 dna modified by n. Structural biochemistryreca protein wikibooks, open books. As anucleoprotein filament, reca promotes aseries of easily monitored dna strand exchange reactions in vitro. Reca is a 38 kilodalton protein essential for the repair and maintenance of dna. Extended active nucleoprotein filaments, in which reca protein is bound to ssdna singlestranded dna in the presence of atp or a nonhydrolyzable atp analog, have a helical diameter of 100 a, a pitch of 95 a, an axial rise per nucleotide of 5. The amino terminal domain of each monomer, consisting of. Transcriptional analysis of the reca gene of streptococcus. Two programmed cell death systems in escherichia coli. Whereas, the binding to singlestranded dna occurred at neutral ph. In order to better understand how sos response modulates swarming, in this work the location of reca and chew proteins within the swarming cells has been studied by using superresolution. May 22, 2008 the reca family of atpases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. The reca protein has alanine and phenylalanine as its nh2 and coohterminal amino acids, respectively, and has the following amino acid composition. Formation of nucleoprotein filaments by dna and reca proteins.