Homologous recombination / DNA repair / phage / Helicase / Nuclease / Inhibitor / Protein complex / Enzyme / DNA mimic / DNA BINDING PROTEIN
Function / homology
Function and homology information
exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA 5'-3' helicase / DNA 3'-5' helicase / recombinational repair / single-stranded DNA helicase activity / 3'-5' DNA helicase activity / DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA 5'-3' helicase / DNA 3'-5' helicase / recombinational repair / single-stranded DNA helicase activity / 3'-5' DNA helicase activity / DNA helicase activity / isomerase activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / 5'-3' DNA helicase activity / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol Similarity search - Function
Biotechnology and Biological Sciences Research Council (BBSRC)
BB/S007261/1
United Kingdom
Medical Research Council (MRC, United Kingdom)
United Kingdom
Citation
Journal: Elife / Year: 2022 Title: Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub. Authors: Martin Wilkinson / Oliver J Wilkinson / Connie Feyerherm / Emma E Fletcher / Dale B Wigley / Mark S Dillingham / Abstract: Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, ...Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.
Name: RecBCD-Abc2-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 Details: All proteins co-expressed and purified as one complex. DNA added prior to making grids in a 1.5x molar excess
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Supramolecule #2: RecBCD enzyme subunit RecB, RecC and RecD
Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.1 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Name
Formula
25.0 mM
Tris-HCl
100.0 mM
Sodium chloride
NaCl
0.5 mM
TCEP
5.0 mM
Magnesium chloride
MgCl2
2.0 mM
ADPNP
Grid
Model: C-flat-2/1 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE Details: Mixture of graphene oxide with 0.3 mM DDM detergent applied directly to grids twice before application of sample
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV / Details: 1.5s blot time.
Details
RecBCD-Abc2 mixed with 1.5x molar excess of synthesised DNA substrate, 2 mM ADPNP and 5 mM MgCl2 prior to making grids
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2500 / Average exposure time: 12.0 sec. / Average electron dose: 56.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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