Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | mechanism of nuclease activation, an alpha-helix (residues 913-922) within the linker region (residues 870-940) of RecB, that connects the C-terminal nuclease domain to the N-terminal helicase domains, sits in the nuclease active site thus blocking access, overview. The nuclease activity of the RecBCD complex is attenuated in the initiation complex prior to binding ATP, because the nuclease requires ssDNA to be fed into it by the helicase activities of the complex | Escherichia coli |
Cloned (Comment) | Organism |
---|---|
genes recBCD, recombinant expression of His-tagged wild-type and mutant D1080A enzyme complexes from three plasmids, pETduet-His6-TEVsite-recBD1080A, pRSFduet-recC and pCDFduet-recD in a DrecBD in Escherichia coli | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme complex RecBCD bound with a DNA substrate (a 350 kDa complex) or ATP analogue ADPNP, mixing of 0.0015 ml of 5 mg/ml protein, and a 1.75fold excess of DNA, 2 mM ADPNP, and 4 mM MgCl2, in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM TCEP, with 0.03 ml of 0.1% detergent n-dodecyl beta-D-maltoside on ice, incubation at 4°C overnight, X-ray diffraction structure determination and analysis at 3.8 A resolution, molecular replacement and modelling using the crystal structure of RecBCD in complex with an extended DNA fork (PDB ID 3K70) as a template | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P08394 AND P07648 AND P04993 | RecBCD enzyme subunits RecB, RecC, and RecD, i.e. beta-, gamma-, and alpha-subunits | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant D1080A enzyme complexes from Escherichia coli by nickel affinity chromatography, heparin affinity chromatography, dialysis, and anion exchange chromatography | Escherichia coli |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
Exonucleolytic cleavage (in the presence of ATP) in either 5'- to 3'- or 3'- to 5'-direction to yield 5'-phosphooligonucleotides | the enzyme complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains | Escherichia coli |
Subunits | Comment | Organism |
---|---|---|
More | structure of the SH3-fold RecD 2B domain, overview | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
RecBCD | - |
Escherichia coli |
RecBCD-type helicase-nuclease | - |
Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | ATP-binding site in the RecB subunit | Escherichia coli | |
additional information | the ATP analogue ADPNP binds in the ATP-binding site of the RecB subunit | Escherichia coli |
General Information | Comment | Organism |
---|---|---|
metabolism | the RecBCD/AddAB complexes are highly processive helicase/nuclease machines that digest DNA from the broken end until they encounter a crossover hotspot instigator (Chi) sequence. At this point their activities are modulated to produce a 3-tailed duplex onto which RecA is loaded | Escherichia coli |
additional information | the SH3 domain interacts with the ssDNA tail in a location different to that normally occupied by a peptide in canonical eukaryotic SH3 domains, thus retaining the potential to bind peptide at the same time as the ssDNA tail, interactions with the 5'-tail and the RecD subunit, overview. The N-terminal domain of RecD contacts the 2B domain of RecC and the conformational change in RecD has significant effects on the conformation of the RecC subunit. The RecB nuclease domain bridges a gap between the 1A and 2B domains of RecC and the conformational changes cause a significant shift in position of the RecB nuclease relative to these domains that reveals the mechanism for nuclease activation. Interaction between the 2B domain of RecD and the RecB subunit has a number of consequences, analysis of conformational changes that proceed from the initiation complex to nuclease activation | Escherichia coli |
physiological function | RecBCD-type helicase-nuclease (RecBCD) catalyses the processing of double-stranded DNA breaks for repair by homologous recombination. The enzyme complex is a highly processive, duplex unwinding and degrading machine that requires tight regulation. The nuclease activity of the complex is activated once unwinding progresses | Escherichia coli |