RecB (PP_4673) is the catalytic helicase-nuclease subunit of the heterotrimeric RecBCD enzyme (Exonuclease V) of Pseudomonas putida KT2440. Its N-terminal UvrD-like superfamily 1 domains form an ATP-dependent 3'-5' DNA helicase/translocase that, together with the oppositely directed RecD motor, unwinds DNA from a double-strand end, and its C-terminal Mg2+-dependent PD-(D/E)XK nuclease domain, the only nuclease active site of the complex, degrades the unwound strands and helps generate a 3' single-stranded tail onto which RecA is loaded, initiating double-strand break repair by homologous recombination. RecC supplies the recognition and scaffold functions. Pseudomonas RecBCD enzymes confer recombination proficiency and ATP-dependent nuclease activity but do not respond to the Escherichia coli Chi octamer; the sequence or structural signal that regulates the KT2440 enzyme is not known. In the related pseudomonad P. syringae, recB null mutants are sensitive to UV and mitomycin C and accumulate fragmented chromosomal DNA, and the RecB ATPase is required in vivo whereas its nuclease is partly dispensable.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | MODIFY | Summary: RecB binds ATP at its Walker A motif (residues 21-28) to power its motor; nucleotide binding is true but is the uninformative parent of ATP binding. Reason: Correct but general. The bound nucleotide is ATP, so the specific descendant GO:0005524 ATP binding, which the gene already carries (accepted here), is the right term. Proposed replacements: ATP binding Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt DR PROSITE; PS51198; UVRD_HELICASE_ATP_BIND; 1. file:PSEPK/recB/recB-uniprot.txt The N-terminal DNA-binding domain is a ssDNA-dependent ATPase and has ATP-dependent 3'-5' helicase function. |
| GO:0000287 magnesium ion binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: The RecB PD-(D/E)XK nuclease domain binds one Mg2+ (D985, D1104, D1117 in Q88DZ5, equivalent to E. coli D956, D1067, D1080), required for nuclease activity. Reason: Correct and as specific as warranted; the HAMAP MF_01485 rule places the conserved metal ligands in this sequence. Cofactor binding supports the core exodeoxyribonuclease V activity rather than being a function in its own right, so non-core (consistent with E. coli RecB). Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt Binds 1 Mg(2+) ion per subunit. file:PSEPK/recB/recB-uniprot.txt DR Pfam; PF12705; PDDEXK_1; 1. |
| GO:0000724 double-strand break repair via homologous recombination | IEA GO_REF:0000104 | ACCEPT | Summary: RecBCD initiates double-strand break repair by homologous recombination. Pseudomonas RecBCD clones restore recombination to an E. coli recBCD deletion, and pseudomonad recB null mutants are DNA-damage sensitive. Reason: Core process. Supported by the HAMAP-curated family function, by P. putida recBCD clones conferring recombination proficiency, and by P. syringae genetics showing all three RecBCD subunits are required for DNA repair. Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt A helicase/nuclease that prepares dsDNA breaks (DSB) for recombinational DNA repair. PMID:2559208 Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. PMID:20195537 All three subunits of the RecBCD(Ps) enzyme are essential for DNA repair and growth of P. syringae at low temperatures |
| GO:0000725 recombinational repair | IEA GO_REF:0000118 | MODIFY | Summary: TreeGrafter transfer of the PAINT PTN000116141 recombinational repair assertion (PTHR11070 UvrD/RecB/PcrA family). Reason: Correct but general. PTN000116141 sets the term at the level appropriate for the whole superfamily 1A family; for RecB the specific child GO:0000724 double-strand break repair via homologous recombination, which the gene already carries (accepted here), is supported. Consistent with the E. coli RecB review. Proposed replacements: double-strand break repair via homologous recombination Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt DR PANTHER; PTHR11070:SF23; RECBCD ENZYME SUBUNIT RECB; 1. PMID:20195537 Among the two major DNA repair pathways (recBCD and recFOR) of bacterial cell, RecBCD machinery is primarily responsible for repairing the double stranded DNA breaks (DSBs). |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: RecB binds DNA as part of the RecBCD end-processing machine. Reason: Correct but uninformative. The biologically relevant binding is to double-strand break ends, so the specific descendant GO:0045027 DNA end binding (definition "Binding to DNA ends exposed by the creation of double-strand breaks (DSBs).") is proposed, as for E. coli RecB. Proposed replacements: DNA end binding Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt Binds to DSBs and unwinds DNA via a highly |
| GO:0003678 DNA helicase activity | IEA GO_REF:0000120 | MODIFY | Summary: RecB is an ATP-dependent DNA helicase of defined 3'-5' polarity. Reason: Correct but general; the specific descendant GO:0043138 3'-5' DNA helicase activity, which the gene already carries (accepted here), is the precise term. Proposed replacements: 3'-5' DNA helicase activity Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt The N-terminal DNA-binding domain is a ssDNA-dependent ATPase and has ATP-dependent 3'-5' helicase function. |
| GO:0004386 helicase activity | IEA GO_REF:0000104 | MODIFY | Summary: Generic helicase activity; RecB is specifically a 3'-5' DNA helicase. Reason: Correct but general; MODIFY to the descendant GO:0043138 3'-5' DNA helicase activity, already carried by the gene. Proposed replacements: 3'-5' DNA helicase activity Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt DR Pfam; PF00580; UvrD-helicase; 1. file:PSEPK/recB/recB-uniprot.txt The N-terminal DNA-binding domain is a ssDNA-dependent ATPase and has ATP-dependent 3'-5' helicase function. |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: RecB carries a UvrD-like ATP-binding domain with a P-loop (residues 21-28) that binds ATP to drive its motor. Reason: Supported by the conserved ATP-binding domain and by pseudomonad genetics showing that the RecB ATP-dependent function is essential in vivo; consistent with E. coli RecB. Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt DR PROSITE; PS51198; UVRD_HELICASE_ATP_BIND; 1. PMID:20195537 while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential |
| GO:0005829 cytosol | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer of the PAINT PTN000869814 cytosol assertion (seeded by E. coli UvrD and Rep). Reason: A reasonable location for a soluble bacterial DNA-repair enzyme acting on the chromosome; Q88DZ5 has no transmembrane segment or signal peptide. There is no localization assay for this protein and the location says little about its function, so non-core (consistent with E. coli RecB). Supporting Evidence: file:PSEPK/recB/recB-deep-research-falcon.md Functionally, RecBCD acts on chromosomal DNA ends and replication-associated DNA structures; thus, the relevant localization is **cytoplasmic/nucleoid-associated**, not membrane-embedded or secreted. |
| GO:0006281 DNA repair | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the RecB family signature; RecB repairs double-strand breaks by homologous recombination. Reason: True but general; MODIFY to the descendant GO:0000724 double-strand break repair via homologous recombination, already carried by the gene. Proposed replacements: double-strand break repair via homologous recombination Supporting Evidence: PMID:20195537 Among the two major DNA repair pathways (recBCD and recFOR) of bacterial cell, RecBCD machinery is primarily responsible for repairing the double stranded DNA breaks (DSBs). |
| GO:0008854 exodeoxyribonuclease V activity | IEA GO_REF:0000120 | ACCEPT | Summary: RecB carries the Exonuclease V (EC 3.1.11.5) nuclease active site of RecBCD in its C-terminal PD-(D/E)XK domain (active site D1117). Reason: Core molecular function. P. putida recBCD clones express ATP-dependent nuclease activity in E. coli, and in P. syringae mutating the equivalent RecB nuclease centre (D1118A) strongly reduces exonuclease activity of the complex. Supporting Evidence: PMID:2559208 Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. file:PSEPK/recB/recB-uniprot.txt The C-terminal domain has nuclease activity and interacts with RecD. |
| GO:0009338 exodeoxyribonuclease V complex | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer of PAINT PTN000116249 (bacterial RecB clade), which is seeded by E. coli RecB. Reason: RecB is an obligate subunit of the RecBCD heterotrimer, and pseudomonad genetics shows that the three subunits act together as a complex. Core. Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt Heterotrimer of RecB, RecC and RecD. PMID:20195537 The RecD requirement is only a function of the RecBCD complex in the bacterium. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000120 | MODIFY | Summary: Very general term; RecB hydrolyses both ATP (motor) and DNA phosphodiester bonds (nuclease). Reason: True but uninformative. Both specific hydrolase activities are carried by the gene and are is_a descendants of hydrolase activity, GO:0016887 ATP hydrolysis activity and GO:0008854 exodeoxyribonuclease V activity. Proposed replacements: ATP hydrolysis activity exodeoxyribonuclease V activity Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt In the holoenzyme this subunit contributes ATPase, 3'-5' helicase, exonuclease activity |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea mapping (RHEA:13065) from the HAMAP-curated ATP hydrolysis reaction of RecB. Reason: RecB is a ssDNA-dependent ATPase, and the term is as precise as the reaction. The ATPase is the energy-transducing component of the coupled 3'-5' helicase captured as core by GO:0043138, so it is kept as non-core (consistent with E. coli RecB). Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt Couples ATP hydrolysis with the unwinding of duplex DNA by translocating in the 3'-5' direction. PMID:20195537 while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential |
| GO:0043138 3'-5' DNA helicase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RecB is the 3'-5' helicase motor of RecBCD, translocating along the 3'-ended strand. Reason: Core molecular function. The 3'-5' polarity is ancestral to the PTHR11070 family (PAINT PTN000116141) and experimentally established for E. coli RecB; Q88DZ5 is in the RecB subfamily and retains the motor domains, and in P. syringae the RecB ATPase-dependent helicase is essential in vivo. Supporting Evidence: file:PSEPK/recB/recB-uniprot.txt The N-terminal DNA-binding domain is a ssDNA-dependent ATPase and has ATP-dependent 3'-5' helicase function. PMID:20195537 is critically dependent on the helicase activities of both RecB and RecD subunits, but not on the nuclease of RecBCD(Ps) enzyme |
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Download this section (compressed HTML)Q: Does KT2440 RecBCD respond to a species-specific sequence or DNA-structure signal, given its failure to recognize the E. coli Chi octamer?
Q: Is the RecB nuclease of KT2440, like that of P. syringae Lz4W, largely dispensable for DNA-damage survival, and does this contribute to the poor homologous recombination efficiency of KT2440?
Experiment: Construct a P. putida KT2440 recB (PP_4673) deletion and complement with wild-type and motor-dead/nuclease-dead alleles to test sensitivity to UV and mitomycin C and quantify homologous recombination capacity.
Experiment: Purify KT2440 RecBCD and map nuclease attenuation and RecA loading on DNA substrates carrying candidate Chi-like octamers to identify the regulatory signal.
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