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Information on EC 3.6.4.B7 - RadA recombinase

for references in articles please use BRENDA:EC3.6.4.B7
preliminary BRENDA-supplied EC number
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UNIPROT: Q55075
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Word Map
The expected taxonomic range for this enzyme is: Archaea, Bacteria, Eukaryota
Reaction Schemes
Synonyms
rad51, rada/sms, ssorada, dna repair protein rad51 homolog 1, rada recombinase, rada intein, radc1, dna repair and recombination protein, mvrada, hvo rada, more
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
involved in DNA repair and in homologous recombination. Binds and assemble on single-stranded DNA to form a nucleoprotein filament. Hydrolyzes ATP in a ssDNA-dependent manner and promotes DNA strand exchange between homologous DNA molecules
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Sulfolobus solfataricus single-stranded DNA binding protein
the tetrameric form of Sulfolobus solfataricus single-stranded DNA binding protein significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Direct interaction between Sulfolobus solfataricus single-stranded DNA binding protein and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Rad54 protein
the Rad54 protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the SsoRadA, to stimulate DNA strand exchange
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ssDNA
the RadA protein is a ssDNA-dependent ATPase
SsoRal1 protein
in addition to constraining SsoRadA ssDNA-dependent ATPase activity, SsoRal1 enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. This results in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggests a filament stabilization function for the SsoRal1 protein
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SsoRal3
UniProtID Q97X93, pI of 6.7, a recombinase paralogue from Sulfolobus solfataricus that enhances SsoRadA ssDNA binding and strand displacement. SsoRal3 protein is a ssDNA-dependent ATPase that can catalyze strand invasion at both saturating and subsaturating concentrations. It can bind both ssDNA and dsDNA, but its binding preference is altered by the presence or absence of ATP. SsoRal3 alters SsoRadA ssDNA-dependent ATPase activity, addition of SsoRal3 to SsoRadA nucleoprotein filaments reduces total ATPase activity. Subsaturating concentrations of SsoRal3 increase the ssDNA binding activity of SsoRadA approximately 9fold and also increase the persistence of SsoRadA catalyzed strand invasion products. SsoRal3 functions to stabilize the SsoRadA presynaptic filament. SsoRal3 can catalyze strand invasion and extends the persistence of SsoRadA-mediated D-loops
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 0.17
ATP
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
displacement loop assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65 - 85
DNA-dependent ATP hydrolysis occurrs exclusively at elevated temperatures (65°C, 75°C, 85°C), and very little hydrolysis occurrs at 37°C
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
i.e. Sulfolobus solfataricus
SwissProt
Manually annotated by BRENDA team
i.e. Sulfolobus solfataricus
SwissProt
Manually annotated by BRENDA team
i.e. Sulfolobus solfataricus
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme belongs to the RecA/RadA family of recombinase proteins of the AAA + ATPases, including RecA proteins of bacteria, RadAs in archaea, Rad51 and DMC1 proteins in Eukaryotes. Archaea and eukaryotes encode RadA/Rad51 paralogues, such as Rad55/57 in yeast, Rad51B/C/D, Xrcc2 and Xrcc3 in mammals, and RadB, RadC in Archaea, which facilitate homologous recombination by interacting with RadA/Rad51 recombinases. Archaeal RadA and eukaryotic Rad51 proteins show high amino acid sequence identities to each other (over 40%) but they are more distantly related to bacterial RecA proteins, exhibiting about 20% sequence identity. Archaeal and eukaryotic recombinases are also more closely related to each other at protein domain structure. RadA paralogues represent another major group of AAA + ATPases involved in DNA damage repair in Archaea
metabolism
nanobiomotors perform various important functions in the cell, and they also emerge as potential vehicle for drug delivery. The proteins employ conserved ATPase domains to convert chemical energy to mechanical work and motion. Some are active during DNA damage repair. All nanobiomotors contain an ATPase domain that adopts RecA fold structure, characteristic for RecA/RadA family proteins, structural analyses of archaeal nucleic acid biomotors and the molecular mechanisms of how ATP binding and hydrolysis promote the conformation change that drives mechanical motion
physiological function
additional information
the ring and right-handed filament structures of RadAs, domain structure and organization, overview. In the absence of ATP, the Walker motifs adopt a conformation to hold the ATP-binding site open and do not interact with the adjacent ATPase domain
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
RADA_SACS2
Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
324
0
35867
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35867
x * 35867, calculated from sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
polymer
the enzyme can selfpolymerize into left-handed helical filaments
additional information
enzyme domain organization, structure comparisons, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, PDB IDs 2DFL and 2BKE
crystal structure of full-length enzyme, quality crystals are grown at 4°C using the hanging drop vapour diffusion method against a reservoir containing 0.5 ml 0.1 M Tris–HCl, pH 9.5 with 10% (v/v) tert-butanol, resolution of 3.2 A. crystal structure reveals a conformation of fine filaments in the absence of nucleotides
crystallized using the hanging drop vapor diffusion method, crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. These five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation
hanging drop vapour diffusion method, crystal structure of the left-handed archaeal RadA helical filament
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K120A
reduced ATPase activity, mutant K120A is able to bind ssDNA with ATP, ADP, or ATPgammaS under saturating protein conditions, but failed to bind well at subsaturating concentrations with ATP or ATPgammaS
K120R
reduced ATPase activity, mutant only binds ATP in the presence of ssDNA
K27R
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
K60R
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, binds dsDNA as well as wild-type protein
R217A
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
R217K
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, mutant exhibits slower ssDNA association rate, surface plasmon resonance binding signals is similar to that of wild-type protein, exhibits weaker affinity to dsDNA as compared to wild-type protein
R223A
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, 90–100% reduction of the surface plasmon resonance binding signal, mutants is defective in dsDNA binding
R223K
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, surface plasmon resonance binding signals is similar to that of wild-type protein, mutants is defective in dsDNA binding
R229A
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, association and dissociation kinetics largely identical or similar to that of the wild-type protein, 90–100% reduction of the surface plasmon resonance binding signal, mutants is defective in dsDNA binding
R229K
mutant does not produced a D-loop product as compared to that of the wild type SsoRadA protein, surface plasmon resonance binding signals is similar to that of wild-type protein, mutants is defective in dsDNA binding
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant proteins K120A and K120R
recombinant enzyme from Escherichia coli strain Rosetta by affinity and anion exchange chromatography, and dialysis, protein concentration using PEG, method, overview
recombinant enzyme from Escherichia coli strain Rosetta by affinity chromatography, dialysis, anion exchange cchromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene radA, recombinant expression in Escherichia coli strain Rosetta
gene radA, recombinant overexpression in Escherichia coli strain Rosetta
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pharmacology
application potential of archaeal nanobiomotors in drug delivery
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Graham, W.J.; Haseltine, C.A.
A recombinase paralog from the hyperthermophilic crenarchaeon Sulfolobus solfataricus enhances SsoRadA ssDNA binding and strand displacement
Gene
515
128-139
2013
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Graham, W.J. 5th; Rolfsmeier, M.L.; Haseltine, C.A.
An archaeal RadA paralog influences presynaptic filament formation
DNA Repair
12
403-413
2013
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Seitz, E.M.; Brockman, J.P.; Sandler, S.J.; Clark, A.J.; Kowalczykowski, S.C.
RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange
Genes Dev.
12
1248-1253
1998
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Rolfsmeier, M.L.; Haseltine, C.A.
The single-stranded DNA binding protein of Sulfolobus solfataricus acts in the presynaptic step of homologous recombination
J. Mol. Biol.
397
31-45
2010
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus
Manually annotated by BRENDA team
Seitz, E.M.; Kowalczykowski, S.C.
The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins
Mol. Microbiol.
37
555-560
2000
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Ariza, A.; Richard, D.J.; White, M.F.; Bond, C.S.
Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA
Nucleic Acids Res.
33
1465-1473
2005
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Chen, L.T.; Ko, T.P.; Chang, Y.C.; Lin, K.A.; Chang, C.S.; Wang, A.H.; Wang, T.F.
Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins
Nucleic Acids Res.
35
1787-1801
2007
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus P2 (Q55075)
Manually annotated by BRENDA team
Haseltine, C.A.; Kowalczykowski, S.C.
An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA
Nucleic Acids Res.
37
2757-2770
2009
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus
Manually annotated by BRENDA team
Chen, L.T.; Ko, T.P.; Chang, Y.W.; Lin, K.A.; Wang, A.H.; Wang, T.F.
Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein
PLoS One
2
e858
2007
Saccharolobus solfataricus (Q55075)
Manually annotated by BRENDA team
Han, W.; Shen, Y.; She, Q.
Nanobiomotors of archaeal DNA repair machineries: current research status and application potential
Cell Biosci.
4
32
2014
Archaeoglobus fulgidus, Sulfolobus islandicus, Sulfurisphaera tokodaii, Methanococcus voltae (O73948), Pyrococcus furiosus (O74036), Saccharolobus solfataricus (Q55075), Thermoplasma acidophilum (Q9HJ68), Saccharolobus solfataricus P2 (Q55075), Thermoplasma acidophilum ATCC 25905 (Q9HJ68)
Manually annotated by BRENDA team
Rolfsmeier, M.; Haseltine, C.
The RadA recombinase and paralogs of the hyperthermophilic archaeon Sulfolobus solfataricus
Methods Enzymol.
600
255-284
2018
Saccharolobus solfataricus (Q55075), Saccharolobus solfataricus, Saccharolobus solfataricus P2 (Q55075), Saccharolobus solfataricus JCM 11322 (Q55075), Saccharolobus solfataricus ATCC 35092 (Q55075), Saccharolobus solfataricus DSM 1617 (Q55075)
Manually annotated by BRENDA team