|
Mode of Action (MoA)
|
mimics DNA and binds to the RecB subunit of RecBCD, competing sterically with DNA. Triggers retron Ec48 defence
|
|
Evidence
|
Gp5.9 was identified as a RecBCD inhibitor through in vitro helicase assays using purified protein. Addition of Gp5.9 to RecBCD prevented DNA unwinding in a double-stranded break resection assay, indicating potent inhibition of RecBCD activity. The specificity of Gp5.9 for RecBCD was demonstrated by its lack of effect on the AddAB complex homologous to RecBCD. A quantitative fluorescence-based helicase assay showed Gp5.9 inhibits RecBCD with an IC50 of 1.3 nM. Electrophoretic mobility shift assays (ECO_0000096) confirmed that Gp5.9 prevents DNA binding by RecBCD, even in complexes already bound by Abc2, indicating a direct competitive mechanism. CryoEM structural analysis of the RecBCD-Gp5.9 complex revealed that Gp5.9 acts as a DNA mimic (ECO_0006181), occupying the DNA binding site on the RecB arm domain and sterically preventing substrate access. This binding mode explains the observed loss of helicase activity and validates Gp5.9’s function as an anti-RecBCD factor that protects phage DNA from degradation.
|
|
MoA Category
|
binds and inhibits host defence system
|
|
Subtype(s) of the defence system(s) inhibited by the protein
Defence Subtype
|
Escherichia coli RecBCD
|
|
Relevant publication(s)
DOI
|
10.7554/eLife.83409
|
|
Other components of the anti-defence system
Multicomponent System
|
-
|
|
Known structure in PDB
PDB ID
|
8B1R_P
|
|
Genome(s) encoding the protein
Protein Source
|
Enterobacteria phage T4
|
|
Defence system(s) inhibited by the protein
Defences
|
RecBCD
|