|Gene:||holB||Accession Numbers: EG11500 (EcoCyc), b1099, ECK1085|
The crystal structure of delta' has been determined. It is composed of three domains arranged in a "C" shape, with a nonfunctional nucleotide binding site in its amino-terminus [Guenther97].
|Map Position: [1,154,985 -> 1,155,989] (24.89 centisomes)||Length: 1005 bp / 334 aa|
Molecular Weight of Polypeptide: 36.937 kD (from nucleotide sequence)
Unification Links: ASAP:ABE-0003716 , CGSC:31447 , DIP:DIP-9932N , EchoBASE:EB1463 , EcoGene:EG11500 , EcoliWiki:b1099 , Mint:MINT-1239429 , ModBase:P28631 , OU-Microarray:b1099 , PortEco:holB , PR:PRO_000022917 , Protein Model Portal:P28631 , RefSeq:NP_415617 , RegulonDB:EG11500 , SMR:P28631 , String:511145.b1099 , UniProt:P28631
Relationship Links: InterPro:IN-FAMILY:IPR004622 , InterPro:IN-FAMILY:IPR008921 , InterPro:IN-FAMILY:IPR015199 , InterPro:IN-FAMILY:IPR027417 , PDB:Structure:1A5T , PDB:Structure:1JR3 , PDB:Structure:1XXH , PDB:Structure:1XXI , PDB:Structure:3GLF , PDB:Structure:3GLG , PDB:Structure:3GLH , PDB:Structure:3GLI , Pfam:IN-FAMILY:PF09115
In Paralogous Gene Group: 134 (5 members)
|Biological Process:||GO:0006261 - DNA-dependent DNA replication
GO:0006260 - DNA replication [UniProtGOA11, GOA01]
GO:0090305 - nucleic acid phosphodiester bond hydrolysis [GOA01]
|Molecular Function:||GO:0005515 - protein binding
[Rajagopala14, Kelman98, Olson95, Simonetta09, Butland05, Kazmirski04, Bullard02, Song01, Jeruzalmi01, Song01a, Glover01, Pritchard00, Gao01c, Li08d]
GO:0003677 - DNA binding [GOA01, Magdalena04]
GO:0003887 - DNA-directed DNA polymerase activity [UniProtGOA11, GOA01a, GOA01]
GO:0008408 - 3'-5' exonuclease activity [GOA01]
GO:0016740 - transferase activity [UniProtGOA11]
GO:0016779 - nucleotidyltransferase activity [UniProtGOA11]
|Cellular Component:||GO:0009360 - DNA polymerase III complex
GO:0005829 - cytosol [DiazMejia09]
|MultiFun Terms:||information transfer → DNA related → DNA replication|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB Lennox||No||37||Aerobic||7||No [Baba06, Comment 1]|
Subunit of: DNA polymerase III, preinitiation complex
Subunit composition of
DNA polymerase III, preinitiation complex = [DnaX]3[HolB][HolA]
DNA polymerase III, τ subunit = DnaX
DNA polymerase III, δ prime subunit = HolB (summary available)
DNA polymerase III, δ subunit = HolA (summary available)
Component of: DNA polymerase III, holoenzyme (extended summary available)
The preinitiation complex binds primed ssDNA and loads the beta processivity clamp. Following this, the DNA polymerase III (pol III) core enzyme, linked by tau, binds, allowing processive DNA polymerization [ODonnell87].
Several of the preinitiation complex subunits are required for binding and initiation of replication. The delta subunit interacts with the primer-template junction, ensuring proper placement of the preinitiation complex at a replication start site [Magdalena04]. The complex binds single-strand binding protein (SSB)-coated DNA with the assistance of associated psi-chi dimers [Fradkin92, Glover98]. The interaction with SSB bound to DNA is a thousand-fold stronger than that with SSB alone; it also enhances the affinity of SSB for DNA, preventing premature dissociation from the initiation site [Glover98, Witte03].
Beta clamps are loaded onto DNA by the preinitiation complex. Contact between the complex and beta is primarily mediated by delta, which can bind to the beta carboxy-termini and remove the beta clamp from DNA in an ATP-independent manner [Leu00, Naktinis95, Indiani03]. Free delta is also present in the cell, possibly acting to remove beta clamps from DNA where no polymerase is present [Leu00]. Preinitiation complex binding to beta does require ATP, suggesting a possible conformational change to reveal delta [Naktinis95]. The delta and delta' subunits themselves undergo only minor conformational changes on ATP binding [Goedken04]. The gamma subunit can also open the beta clamp, though less effectively than delta [Leu01]. Both gamma and delta' restrict opening of beta by delta in the absence of ATP [Leu01]. The preinitiation complex loads beta onto template DNA at a rate of 12/s, which is fast enough to account for the rate of lagging strand initiation [Bloom96].
Beta loading and preinitiation complex release from DNA are both ATP-dependent processes. Each gamma subunit binds an ATP, prompting a conformational change that induces delta' to permit delta to bind and act on the beta clamp [Hingorani98, Turner99]. ATP binding also raises the affinity of the preinitiation complex for primed template [Ason00]. This same primed template triggers hydrolysis of bound ATP, possibly via the interaction between beta and primed DNA, reverting the complex to a lower affinity form that cycles off the DNA and disengages from beta [Ason03, Turner99, Bertram98]. The involvement of ATP and its hydrolysis in beta loading and subsequent preinitiation complex release has been examined in detail [Hingorani99, Bertram00, Snyder04, Williams04].
A number of structural studies have been carried out on the preinitiation complex, sometimes with conflicting results. In addition to the 2 gamma, 1 delta, 1 delta', 1 chi, 1 psi structure found in the holoenzyme, a tau variant with tau, delta, delta', chi and psi can form in vitro [Onrust95, Onrust95a]. In sedimentation assays, gamma and tau have both been detected as homotetramers both on their own and complexed with delta, delta', chi and psi [Dallmann95]. Within the preinitiation complex, gamma crosslinks with both delta' and psi [Glover00]. A crystal structure of the entire complex at 2.7 A resolution shows a pentameric arrangement of subunits including three gamma subunits [Jeruzalmi01]. Another crystal structure, of delta and beta together, shows that delta operates to open beta by perturbing one of the beta dimer interface points [Jeruzalmi01a].
Subunit of: DNA polymerase III, holoenzyme
Subunit composition of
DNA polymerase III, holoenzyme = [(DnaE)(DnaQ)(HolE)]3[(DnaX)3(HolB)(HolA)][(DnaN)2]2[(DnaX)2][(HolC)(HolD)]4
DNA polymerase III, core enzyme = (DnaE)(DnaQ)(HolE) (summary available)
DNA polymerase III, α subunit = DnaE (extended summary available)
DNA polymerase III, ε subunit = DnaQ (extended summary available)
DNA polymerase III, θ subunit = HolE (extended summary available)
DNA polymerase III, preinitiation complex = (DnaX)3(HolB)(HolA) (extended summary available)
DNA polymerase III, τ subunit = DnaX
DNA polymerase III, δ prime subunit = HolB (summary available)
DNA polymerase III, δ subunit = HolA (summary available)
DNA polymerase III, β subunit = (DnaN)2 (extended summary available)
DNA polymerase III, τ subunit dimer = (DnaX)2 (extended summary available)
DNA polymerase III, τ subunit = DnaX
DNA polymerase III, ψ-χ subunit = (HolC)(HolD) (extended summary available)
DNA polymerase III, χ subunit = HolC
DNA polymerase III, ψ subunit = HolD
DNA polymerase III holoenzyme is the enzyme primarily responsible for replicative DNA synthesis in E. coli. It carries out primer-initiated 5' to 3' polymerization of DNA on a single-stranded DNA template, as well as 3' to 5' exonucleolytic editing of mispaired nucleotides.
Replicative DNA polymerization begins when the preinitiation complex binds single-stranded DNA near an RNA primer. The preinitiation complex then loads the beta processivity clamp onto the DNA at this site, after which three core polymerases, chaperoned into place by the tau subunit, bind to the processivity clamp, with one polymerase on the leading strand and two on the lagging. DNA is synthesized 5' to 3' from primers on both the leading and lagging strands, covalently attaching the newly synthesized DNA to the primer. Tau displaces beta in the presence of duplex DNA, dissociating the polymerase from the template when it reaches a temporary stop on the lagging strand or when synthesis is complete on either strand [Maki88, Maki88a, Onrust95, Maki88b, Nusslein76, ReyesLamothe10].
For more detailed discussion of the stages of polymerase binding and DNA synthesis, see the individual entries for DNA polymerase III, preinitiation complex, DNA polymerase III, β subunit, DNA polymerase III, τ subunit dimer, DNA polymerase III, core enzyme and their constituent parts.
DNA polymerase III binds a region about 30 nucleotides long upstream of the RNA primer, with the alpha subunit making contact 9 nucleotides upstream and the beta clamp making contact 22 nucleotides upstream [Reems95, Reems94]. The preinitiation complex binds an area larger than this prior to being displaced by the core polymerase [Reems94]. In the presence of DNA polymerase III, RNA primer length is limited to 10 nucleotides, a limitation that is independent of the epsilon-mediated 3' to 5' exonuclease activity [Zechner92].
During polymerization, DNA polymerase III pauses at sites of potential secondary structure [LaDuca83]. The holoenzyme can traverse distances as long as 400 base pairs of duplex DNA to reach the next available 3' end and restart synthesis [ODonnell85]. Such jumps within the same template take 2 to 5 seconds, whereas transfer to a new template takes 30 seconds [Burgers83].
DNA polymerase III is required for several kinds of DNA repair, including some forms of double-strand break repair, fixing hydrogen-peroxide-induced damage and methyl-directed mismatch repair [Motamedi99, Hagensee89, Cooper93]. Mutations that inhibit polymerase III stimulate repeat expansion and lead to lower levels of unsaturation in fatty acids [Morag99, Suzuki98].
UV mutagenesis and gap repair following UV damage to DNA both involve DNA polymerase III [CohenFix94, Tomer96]. The polymerase stalls at pyrimidine photodimers in vitro, but is capable of bypassing such lesions to continue synthesis [Shwartz87, Livneh86]. This bypass activity is stimulated by single-strand binding protein (SSB) but inhibited by the polymerase III beta subunit, which explains the relatively low rate of bypass in vivo [Shwartz87, Shavitt89].
10/20/97 Gene b1099 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11500; confirmed by SwissProt match.
Ason00: Ason B, Bertram JG, Hingorani MM, Beechem JM, O'Donnell M, Goodman MF, Bloom LB (2000). "A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader." J Biol Chem 275(4);3006-15. PMID: 10644772
Ason03: Ason B, Handayani R, Williams CR, Bertram JG, Hingorani MM, O'Donnell M, Goodman MF, Bloom LB (2003). "Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp." J Biol Chem 278(12);10033-40. PMID: 12519754
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
Bertram00: Bertram JG, Bloom LB, Hingorani MM, Beechem JM, O'Donnell M, Goodman MF (2000). "Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA." J Biol Chem 275(37);28413-20. PMID: 10874049
Bertram98: Bertram JG, Bloom LB, Turner J, O'Donnell M, Beechem JM, Goodman MF (1998). "Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA." J Biol Chem 273(38);24564-74. PMID: 9733751
Bloom96: Bloom LB, Turner J, Kelman Z, Beechem JM, O'Donnell M, Goodman MF (1996). "Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA." J Biol Chem 271(48);30699-708. PMID: 8940047
Bullard02: Bullard JM, Pritchard AE, Song MS, Glover BP, Wieczorek A, Chen J, Janjic N, McHenry CS (2002). "A three-domain structure for the delta subunit of the DNA polymerase III holoenzyme delta domain III binds delta' and assembles into the DnaX complex." J Biol Chem 277(15);13246-56. PMID: 11809766
Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043
Carter93: Carter JR, Franden MA, Aebersold R, McHenry CS (1993). "Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme." J Bacteriol 175(12);3812-22. PMID: 8509334
Dallmann95: Dallmann HG, McHenry CS (1995). "DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Physical characterization of the DnaX subunits and complexes." J Biol Chem 270(49);29563-9. PMID: 7493999
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
Gao01c: Gao D, McHenry CS (2001). "Tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain III, shared by gamma and tau, binds delta delta ' and chi psi." J Biol Chem 276(6);4447-53. PMID: 11078742
Glover01: Glover BP, Pritchard AE, McHenry CS (2001). "tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX." J Biol Chem 276(38);35842-6. PMID: 11463787
Glover98: Glover BP, McHenry CS (1998). "The chi psi subunits of DNA polymerase III holoenzyme bind to single-stranded DNA-binding protein (SSB) and facilitate replication of an SSB-coated template." J Biol Chem 273(36);23476-84. PMID: 9722585
Goedken04: Goedken ER, Levitus M, Johnson A, Bustamante C, O'Donnell M, Kuriyan J (2004). "Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding." J Mol Biol 336(5);1047-59. PMID: 15037068
Guenther97: Guenther B, Onrust R, Sali A, O'Donnell M, Kuriyan J (1997). "Crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III." Cell 91(3);335-45. PMID: 9363942
Hingorani98: Hingorani MM, O'Donnell M (1998). "ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme." J Biol Chem 273(38);24550-63. PMID: 9733750
Hingorani99: Hingorani MM, Bloom LB, Goodman MF, O'Donnell M (1999). "Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA." EMBO J 18(18);5131-44. PMID: 10487764
Jeruzalmi01a: Jeruzalmi D, Yurieva O, Zhao Y, Young M, Stewart J, Hingorani M, O'Donnell M, Kuriyan J (2001). "Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III." Cell 106(4);417-28. PMID: 11525728
Kazmirski04: Kazmirski SL, Podobnik M, Weitze TF, O'Donnell M, Kuriyan J (2004). "Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex." Proc Natl Acad Sci U S A 101(48);16750-5. PMID: 15556993
Kelman98: Kelman Z, Yuzhakov A, Andjelkovic J, O'Donnell M (1998). "Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly." EMBO J 17(8);2436-49. PMID: 9545254
LaDuca83: LaDuca RJ, Fay PJ, Chuang C, McHenry CS, Bambara RA (1983). "Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III." Biochemistry 22(22);5177-88. PMID: 6360204
Leu00: Leu FP, Hingorani MM, Turner J, O'Donnell M (2000). "The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli." J Biol Chem 275(44);34609-18. PMID: 10924523
Leu01: Leu FP, O'Donnell M (2001). "Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme." J Biol Chem 276(50);47185-94. PMID: 11572866
Li08d: Li F, Liu Q, Chen YY, Yu ZN, Zhang ZP, Zhou YF, Deng JY, Bi LJ, Zhang XE (2008). "Escherichia coli mismatch repair protein MutL interacts with the clamp loader subunits of DNA polymerase III." Mutat Res 637(1-2);101-10. PMID: 17765269
Livneh86: Livneh Z (1986). "Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis." J Biol Chem 261(20);9526-33. PMID: 2941423
Magdalena04: Magdalena Coman M, Jin M, Ceapa R, Finkelstein J, O'Donnell M, Chait BT, Hingorani MM (2004). "Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit." J Mol Biol 342(5);1457-69. PMID: 15364574
Maki88: Maki S, Kornberg A (1988). "DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits." J Biol Chem 263(14);6561-9. PMID: 3283127
Maki88a: Maki S, Kornberg A (1988). "DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis." J Biol Chem 263(14);6555-60. PMID: 3283126
Maki88b: Maki H, Maki S, Kornberg A (1988). "DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites." J Biol Chem 263(14);6570-8. PMID: 3283128
Motamedi99: Motamedi MR, Szigety SK, Rosenberg SM (1999). "Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo." Genes Dev 13(21);2889-903. PMID: 10557215
Naktinis95: Naktinis V, Onrust R, Fang L, O'Donnell M (1995). "Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp." J Biol Chem 270(22);13358-65. PMID: 7768937
Nusslein76: Nusslein V, Henke S, Johnston LH (1976). "Replication of E. coli duplex DNA in vitro. The separation of the DNA containing fractions of a lysate from the soluble enzymes and their complementation properties." Mol Gen Genet 145(2);183-90. PMID: 778584
ODonnell87: O'Donnell ME (1987). "Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli." J Biol Chem 262(34);16558-65. PMID: 3316222
Olson95: Olson MW, Dallmann HG, McHenry CS (1995). "DnaX complex of Escherichia coli DNA polymerase III holoenzyme. The chi psi complex functions by increasing the affinity of tau and gamma for delta.delta' to a physiologically relevant range." J Biol Chem 270(49);29570-7. PMID: 7494000
Onrust95: Onrust R, Finkelstein J, Turner J, Naktinis V, O'Donnell M (1995). "Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. III. Interface between two polymerases and the clamp loader." J Biol Chem 270(22);13366-77. PMID: 7768938
Onrust95a: Onrust R, Finkelstein J, Naktinis V, Turner J, Fang L, O'Donnell M (1995). "Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. I. Organization of the clamp loader." J Biol Chem 270(22);13348-57. PMID: 7768936
Pritchard00: Pritchard AE, Dallmann HG, Glover BP, McHenry CS (2000). "A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'." EMBO J 19(23);6536-45. PMID: 11101526
Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554
Reems95: Reems JA, Wood S, McHenry CS (1995). "Escherichia coli DNA polymerase III holoenzyme subunits alpha, beta, and gamma directly contact the primer-template." J Biol Chem 270(10);5606-13. PMID: 7890680
Shavitt89: Shavitt O, Livneh Z (1989). "The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli." J Biol Chem 264(19);11275-81. PMID: 2661556
Shwartz87: Shwartz H, Livneh Z (1987). "Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli." J Biol Chem 262(22);10518-23. PMID: 2956258
Simonetta09: Simonetta KR, Kazmirski SL, Goedken ER, Cantor AJ, Kelch BA, McNally R, Seyedin SN, Makino DL, O'Donnell M, Kuriyan J (2009). "The mechanism of ATP-dependent primer-template recognition by a clamp loader complex." Cell 137(4);659-71. PMID: 19450514
Snyder04: Snyder AK, Williams CR, Johnson A, O'Donnell M, Bloom LB (2004). "Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II. Uncoupling the beta and DNA binding activities of the gamma complex." J Biol Chem 279(6);4386-93. PMID: 14610068
Song01: Song MS, McHenry CS (2001). "Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly." J Biol Chem 276(52);48709-15. PMID: 11606586
Suzuki98: Suzuki E, Kondo T, Makise M, Mima S, Sakamoto K, Tsuchiya T, Mizushima T (1998). "Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication." Biol Pharm Bull 21(7);657-61. PMID: 9703244
Tomer96: Tomer G, Cohen-Fix O, O'Donnell M, Goodman M, Livneh Z (1996). "Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II." Proc Natl Acad Sci U S A 93(4);1376-80. PMID: 8643639
Williams04: Williams CR, Snyder AK, Kuzmic P, O'Donnell M, Bloom LB (2004). "Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex." J Biol Chem 279(6);4376-85. PMID: 14610067
Witte03: Witte G, Urbanke C, Curth U (2003). "DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery." Nucleic Acids Res 31(15);4434-40. PMID: 12888503
Zechner92: Zechner EL, Wu CA, Marians KJ (1992). "Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. III. A polymerase-primase interaction governs primer size." J Biol Chem 267(6);4054-63. PMID: 1531480
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