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Escherichia coli K-12 substr. MG1655 Enzyme: McrBC restriction endonuclease

Subunit composition of McrBC restriction endonuclease = [McrB][McrC]
         MrcB subunit of 5-methylcytosine restriction system = McrB (summary available)
         MrcC subunit of 5-methylcytosine restriction system = McrC (summary available)

Summary:
McrBC is a type IV deoxyribonuclease which is one of three restriction systems defending the cell against foreign DNA, such as bacteriophages. It recognizes DNA containing two methylated cytosine residues separated by 30-2000 base pairs and cleaves the DNA at multiple sites close to one of the methylated sites. McrBC forms high molecular mass complexes with DNA [Panne01, Pieper02].

Restriction of methylated DNA is responsible for difficulties with cloning methylcytosine-containing DNA from various sources in E. coli [Raleigh88, Whittaker88, Kretz89].

Review: [Raleigh92]

Gene-Reaction Schematic: ?


Enzymatic reaction of: McrBC restriction endonuclease

EC Number: 3.1.21.-

DNA containing a recognition site + H2O <=> Cleaved DNA

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is irreversible in the direction shown.

Cofactors or Prosthetic Groups: Mg2+ , GTP [Sutherland92]

Inhibitors (Unknown Mechanism): ATP [Sutherland92]


Subunit of McrBC restriction endonuclease: MrcB subunit of 5-methylcytosine restriction system

Synonyms: RglB, McrB

Gene: mcrB Accession Numbers: EG10574 (EcoCyc), b4346, ECK4336

Locations: cytosol

Sequence Length: 459 AAs

Molecular Weight: 53.157 kD (from nucleotide sequence)

GO Terms:

Biological Process: GO:0000737 - DNA catabolic process, endonucleolytic Inferred from experiment [Ross89]
GO:0006184 - GTP catabolic process Inferred from experiment [Sutherland92]
GO:0006308 - DNA catabolic process Inferred from experiment [Ross89]
GO:0006200 - ATP catabolic process Inferred by computational analysis [GOA01]
GO:0009307 - DNA restriction-modification system Inferred by computational analysis [UniProtGOA11]
GO:0090305 - nucleic acid phosphodiester bond hydrolysis Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0003677 - DNA binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, Kruger95]
GO:0003924 - GTPase activity Inferred from experiment [Sutherland92]
GO:0015666 - restriction endodeoxyribonuclease activity Inferred from experiment [Ross89]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0004518 - nuclease activity Inferred by computational analysis [UniProtGOA11]
GO:0004519 - endonuclease activity Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [GOA01]
GO:0005525 - GTP binding Inferred by computational analysis [UniProtGOA11]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11]
GO:0016887 - ATPase activity Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol

MultiFun Terms: information transfer DNA related DNA degradation
information transfer DNA related DNA restriction/modification
metabolism degradation of macromolecules DNA

Unification Links: DIP:DIP-10168N , EcoliWiki:b4346 , Mint:MINT-1286238 , ModBase:P15005 , PR:PRO_000023171 , Pride:P15005 , Protein Model Portal:P15005 , RefSeq:NP_418766 , SMR:P15005 , String:511145.b4346 , UniProt:P15005

Relationship Links: InterPro:IN-FAMILY:IPR003593 , InterPro:IN-FAMILY:IPR011704 , InterPro:IN-FAMILY:IPR021961 , InterPro:IN-FAMILY:IPR027417 , PDB:Structure:3SSC , PDB:Structure:3SSD , PDB:Structure:3SSE , Pfam:IN-FAMILY:PF07728 , Pfam:IN-FAMILY:PF12102 , Smart:IN-FAMILY:SM00382

Summary:
The McrB protein together with McrC forms the McrBC restriction system [Ross89]. The MrcB subunit mediates binding of the enzyme to hemi- and fully methylated 5'PuC sites [Kruger95]. Sequence determinants for DNA binding have been studied [Sutherland92, Gast97, Stewart]. McrB also binds and hydrolyzes the essential cofactor GTP [Kruger95, Pieper97].

Two isoforms of McrB are produced by alternative translation initiation [Ross89a, Zheng92a]. McrBs, the shorter isoform, was suggested to bind to McrC and thus modulate McrBC activity [Panne98], or to stabilize the McrBC complex [Beary97].

mcrB = "modified cytosine restriction" [Raleigh86]

Essentiality data for mcrB knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Subunit of McrBC restriction endonuclease: MrcC subunit of 5-methylcytosine restriction system

Synonyms: McrC

Gene: mcrC Accession Numbers: EG10575 (EcoCyc), b4345, ECK4335

Locations: cytosol

Sequence Length: 348 AAs

Molecular Weight: 40.59 kD (from nucleotide sequence)

GO Terms:

Biological Process: GO:0009307 - DNA restriction-modification system Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: information transfer DNA related DNA degradation
information transfer DNA related DNA restriction/modification
metabolism degradation of macromolecules DNA

Unification Links: EcoliWiki:b4345 , PR:PRO_000023172 , Protein Model Portal:P15006 , RefSeq:NP_418765 , String:511145.b4345 , UniProt:P15006

Relationship Links: InterPro:IN-FAMILY:IPR014407 , InterPro:IN-FAMILY:IPR019292 , Pfam:IN-FAMILY:PF10117

Summary:
McrC modulates the specificity of McrB [Dila90] and stimulates hydrolysis of GTP by McrB 30-fold [Pieper97]. Site-directed mutagenesis of proposed active site residues has shown that the McrC protein harbors the DNA cleavage activity of the McrBC restriction endonuclease [Pieper02a].

mcrC = "modified cytosine restriction" [Raleigh86]

Essentiality data for mcrC knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Beary97: Beary TP, Braymer HD, Achberger EC (1997). "Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12." J Bacteriol 179(24);7768-75. PMID: 9401036

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dila90: Dila D, Sutherland E, Moran L, Slatko B, Raleigh EA (1990). "Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12." J Bacteriol 172(9);4888-900. PMID: 2203735

Gast97: Gast FU, Brinkmann T, Pieper U, Kruger T, Noyer-Weidner M, Pingoud A (1997). "The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB." Biol Chem 378(9);975-82. PMID: 9348106

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kretz89: Kretz PL, Reid CH, Greener A, Short JM (1989). "Effect of lambda packaging extract mcr restriction activity on DNA cloning." Nucleic Acids Res 17(13);5409. PMID: 2548161

Kruger95: Kruger T, Wild C, Noyer-Weidner M (1995). "McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues." EMBO J 14(11);2661-9. PMID: 7781618

Panne01: Panne D, Muller SA, Wirtz S, Engel A, Bickle TA (2001). "The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides." EMBO J 20(12);3210-7. PMID: 11406597

Panne98: Panne D, Raleigh EA, Bickle TA (1998). "McrBs, a modulator peptide for McrBC activity." EMBO J 17(18);5477-83. PMID: 9736625

Pieper02: Pieper U, Groll DH, Wunsch S, Gast FU, Speck C, Mucke N, Pingoud A (2002). "The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat." Biochemistry 41(16);5245-54. PMID: 11955074

Pieper02a: Pieper U, Pingoud A (2002). "A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli." Biochemistry 41(16);5236-44. PMID: 11955073

Pieper97: Pieper U, Brinkmann T, Kruger T, Noyer-Weidner M, Pingoud A (1997). "Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP." J Mol Biol 272(2);190-9. PMID: 9299347

Raleigh86: Raleigh EA, Wilson G (1986). "Escherichia coli K-12 restricts DNA containing 5-methylcytosine." Proc Natl Acad Sci U S A 83(23);9070-4. PMID: 3024165

Raleigh88: Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PW, Elhai J, Hanahan D (1988). "McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning." Nucleic Acids Res 16(4);1563-75. PMID: 2831502

Raleigh92: Raleigh EA (1992). "Organization and function of the mcrBC genes of Escherichia coli K-12." Mol Microbiol 6(9);1079-86. PMID: 1316984

Ross89: Ross TK, Achberger EC, Braymer HD (1989). "Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12." Mol Gen Genet 216(2-3);402-7. PMID: 2664457

Ross89a: Ross TK, Achberger EC, Braymer HD (1989). "Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus." J Bacteriol 171(4);1974-81. PMID: 2649480

Stewart: Stewart FJ, Raleigh EA "Dependence of McrBC cleavage on distance between recognition elements." Biol Chem 379(4-5);611-6. PMID: 9628366

Sutherland92: Sutherland E, Coe L, Raleigh EA (1992). "McrBC: a multisubunit GTP-dependent restriction endonuclease." J Mol Biol 225(2);327-48. PMID: 1317461

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Whittaker88: Whittaker PA, Campbell AJ, Southern EM, Murray NE (1988). "Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector." Nucleic Acids Res 16(14B);6725-36. PMID: 2841642

Zheng92a: Zheng L, Wang X, Braymer HD (1992). "Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12." Gene 112(1);97-100. PMID: 1312983


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Wed Dec 17, 2014, biocyc12.