Literature summary for 3.1.11.5 extracted from

Wilkinson, M.; Chaban, Y.; Wigley, D.B.
Mechanism for nuclease regulation in RecBCD (2016), eLife, 5, e18227 .

Activating Compound

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(Commentary)

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 (Commentary)

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

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

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Organism

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 (Commentary)

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

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	a

Subunits

Subunits	Comment	Organism
More	structure of the SH3-fold RecD 2B domain, overview	Escherichia coli

Synonyms

Synonyms	Comment	Organism
RecBCD	-	Escherichia coli
RecBCD-type helicase-nuclease	-	Escherichia coli

Cofactor

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

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