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Escherichia coli K-12 substr. MG1655 Enzyme: endonuclease VIII



Gene: nei Accession Numbers: G6383 (EcoCyc), b0714, ECK0703

Regulation Summary Diagram: ?

Summary:
Damaged bases in DNA occur due to alkylating agents, ionizing radiation or oxidants such as superoxide radicals, hydrogen peroxide and hydroxyl radicals. In Escherichia coli, base excision repair (BER) works to repair damaged DNA by multiple steps: removal of the damaged base by a cleavage of the glycosidic bond by a glycosylase enzyme generating an apyrimidinic or apurinic (AP) site; removal of the AP site by an AP endonuclease; resynthesis of the excised strand by DNA polymerase using the intact strand as template and ligation by DNA ligase. In E.coli endonuclease VIII (the nei gene product) functions similarly to DNA glycosylase Nth (the nth gene product) and also releases damaged T and C residues [Jiang97b]. Studies also show that DNA containing apyrimidinic or apurinic (AP) sites is also degraded by the enzyme, suggesting that, in addition to a DNA glycosylase, Nei also functions as an AP lyase [Jiang97a]. The crystal structure of the uncomplexed form of endonuclease VIII (Nei) has been determined to 2.6A resolution and the structures of two Nei active-site specific mutants, R252A and E2A, were also obtained at 2.25 and 2.05 A resolutions, respectively [Golan04].

Gene Citations: [Gifford00]

Locations: cytosol

Map Position: [745,158 -> 745,949] (16.06 centisomes)
Length: 792 bp / 263 aa

Molecular Weight of Polypeptide: 29.845 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0002433 , DIP:DIP-10327N , DisProt:DP00375 , EchoBASE:EB3026 , EcoGene:EG13237 , EcoliWiki:b0714 , Mint:MINT-1221359 , ModBase:P50465 , OU-Microarray:b0714 , PortEco:nei , PR:PRO_000023368 , Protein Model Portal:P50465 , RefSeq:NP_415242 , RegulonDB:G6383 , SMR:P50465 , String:511145.b0714 , UniProt:P50465

Relationship Links: InterPro:IN-FAMILY:IPR000214 , InterPro:IN-FAMILY:IPR010663 , InterPro:IN-FAMILY:IPR010979 , InterPro:IN-FAMILY:IPR012319 , InterPro:IN-FAMILY:IPR015886 , InterPro:IN-FAMILY:IPR015887 , InterPro:IN-FAMILY:IPR023713 , PDB:Structure:1K3W , PDB:Structure:1K3X , PDB:Structure:1Q39 , PDB:Structure:1Q3B , PDB:Structure:1Q3C , PDB:Structure:2EA0 , PDB:Structure:2OPF , PDB:Structure:2OQ4 , Pfam:IN-FAMILY:PF01149 , Pfam:IN-FAMILY:PF06827 , Pfam:IN-FAMILY:PF06831 , Prosite:IN-FAMILY:PS01242 , Prosite:IN-FAMILY:PS51066 , Prosite:IN-FAMILY:PS51068 , Smart:IN-FAMILY:SM00898

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0000737 - DNA catabolic process, endonucleolytic Inferred by computational analysis Inferred from experiment [Kropachev06, Hazra00, GOA06, GOA01, GOA01a]
GO:0006284 - base-excision repair Inferred from experiment Inferred by computational analysis [GOA01a, Kropachev06]
GO:0006281 - DNA repair Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
GO:0006289 - nucleotide-excision repair Inferred by computational analysis [GOA01a]
GO:0006974 - cellular response to DNA damage stimulus Inferred by computational analysis [UniProtGOA11a]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0090305 - nucleic acid phosphodiester bond hydrolysis Inferred by computational analysis [GOA01a]
Molecular Function: GO:0003684 - damaged DNA binding Inferred from experiment Inferred by computational analysis [GOA01a, Kropachev06, Hazra00]
GO:0003906 - DNA-(apurinic or apyrimidinic site) lyase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Kropachev06, Hazra00]
GO:0008270 - zinc ion binding Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Golan05]
GO:0000703 - oxidized pyrimidine nucleobase lesion DNA N-glycosylase activity Inferred by computational analysis [GOA06]
GO:0003676 - nucleic acid binding Inferred by computational analysis [GOA01a]
GO:0003677 - DNA binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0004519 - endonuclease activity Inferred by computational analysis [GOA01a]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016798 - hydrolase activity, acting on glycosyl bonds Inferred by computational analysis [UniProtGOA11a]
GO:0016799 - hydrolase activity, hydrolyzing N-glycosyl compounds Inferred by computational analysis [GOA01a]
GO:0016829 - lyase activity Inferred by computational analysis [UniProtGOA11a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005737 - cytoplasm
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: information transfer DNA related DNA repair

Essentiality data for nei knockouts: ?

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

Enzymatic reaction of: endonuclease

Synonyms: AP-lyase, DNA-(apurinic or apyrimidinic site) lyase

EC Number: 4.2.99.18

a DNA containing an apurinic/apyrimidinic site <=> a 5'-phosphopolynucleotide + a 3'-terminal unsaturated sugar

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

Summary:
In E.coli endonuclease VIII (the nei gene product) functions similarly to DNA glycosylase Nth (the nth gene product) and also releases damaged T and C residues [Jiang97b].


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Jiang97b, UniProt11]
UniProt: Removed.
Mutagenesis-Variant 2
[Burgess02, UniProt11]
Alternate sequence: P → T; UniProt: Loss of glycosylase and AP lyase activity.
Active-Site 2
[UniProt10a]
UniProt: Schiff-base intermediate with DNA;
Chain 2 -> 263
[UniProt09]
UniProt: Endonuclease VIII;
Mutagenesis-Variant 3
[Zharkov02, Burgess02, UniProt11]
Alternate sequence: E → D; UniProt: Much reduced glycosylase activity. No effect on AP lyase activity.
Alternate sequence: E → Q; UniProt: Loss of glycosylase activity. No effect on AP lyase activity.
Alternate sequence: E → A; UniProt: Loss of glycosylase activity. No effect on AP lyase activity.
Active-Site 3
[UniProt10]
UniProt: Proton donor; Non-Experimental Qualifier: probable;
Mutagenesis-Variant 6
[Burgess02, UniProt11]
Alternate sequence: E → Q; UniProt: Reduced glycosylase activity. No effect on AP lyase activity.
Mutagenesis-Variant 53
[Zharkov02, UniProt11]
Alternate sequence: K → A; UniProt: Loss of DNA cleavage at sites containing oxidized pyrimidine. No effect on AP lyase activity.
Active-Site 53
[UniProt10]
UniProt: Proton donor; for beta-elimination activity; Non-Experimental Qualifier: probable;
Amino-Acid-Sites-That-Bind 70
[UniProt10a]
UniProt: DNA;
Amino-Acid-Sites-That-Bind 125
[UniProt10a]
UniProt: DNA;
Mutagenesis-Variant 129
[Burgess02, UniProt11]
Alternate sequence: D → N; UniProt: Reduced glycosylase activity. No effect on AP lyase activity.
Mutagenesis-Variant 160
[Burgess02, UniProt11]
Alternate sequence: D → N; UniProt: No effect on glycosylase and AP lyase activity.
Amino-Acid-Sites-That-Bind 169
[UniProt10a]
UniProt: DNA;
Mutagenesis-Variant 174
[Burgess02, UniProt11]
Alternate sequence: E → Q; UniProt: Loss of glycosylase activity. No effect on AP lyase activity.
Mutagenesis-Variant 213
[Zharkov02, UniProt11]
Alternate sequence: R → A; UniProt: Slightly reduced activity.
Zn-Finger-Region 229 -> 263
[UniProt10a]
UniProt: FPG-type;
Mutagenesis-Variant 253
[Zharkov02, UniProt11]
Alternate sequence: R → A; UniProt: Reduced DNA cleavage at sites containing oxidized pyrimidine. No effect on AP lyase activity.
Active-Site 253
[UniProt10]
UniProt: Proton donor; for delta-elimination activity; Non-Experimental Qualifier: probable;


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


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

Burgess02: Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS (2002). "Determination of active site residues in Escherichia coli endonuclease VIII." J Biol Chem 277(4);2938-44. PMID: 11711552

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

Gifford00: Gifford CM, Wallace SS (2000). "The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons." Nucleic Acids Res 28(3);762-9. PMID: 10637328

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Golan04: Golan G, Zharkov DO, Fernandes AS, Zaika E, Kycia JH, Wawrzak Z, Grollman AP, Shoham G (2004). "Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed form." Acta Crystallogr D Biol Crystallogr 60(Pt 8);1476-80. PMID: 15272182

Golan05: Golan G, Zharkov DO, Feinberg H, Fernandes AS, Zaika EI, Kycia JH, Grollman AP, Shoham G (2005). "Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility." Nucleic Acids Res 33(15);5006-16. PMID: 16145054

Hazra00: Hazra TK, Izumi T, Venkataraman R, Kow YW, Dizdaroglu M, Mitra S (2000). "Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII." J Biol Chem 275(36);27762-7. PMID: 10862773

Jiang97a: Jiang D, Hatahet Z, Melamede RJ, Kow YW, Wallace SS (1997). "Characterization of Escherichia coli endonuclease VIII." J Biol Chem 272(51);32230-9. PMID: 9405426

Jiang97b: Jiang D, Hatahet Z, Blaisdell JO, Melamede RJ, Wallace SS (1997). "Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants." J Bacteriol 179(11);3773-82. PMID: 9171429

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

Kropachev06: Kropachev KY, Zharkov DO, Grollman AP (2006). "Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger." Biochemistry 45(39);12039-49. PMID: 17002303

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Zharkov02: Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G (2002). "Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate." EMBO J 21(4);789-800. PMID: 11847126


Report Errors or Provide Feedback
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 Sat Dec 20, 2014, biocyc12.