Model for the Catalytic Domain of the Proofreading Subunit of Escherichia coli DNA Polymerase III Based on NMR Structural
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- 作者：Eugene F. DeRose ; Dawei Li ; Thomas Darden ; Scott Harvey ; Fred W. Perrino ; Roel M. Schaaper ; and Robert E. London
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 8, 2002
- 年：2002
- 卷：41
- 期：1
- 页码：94 - 110
- 全文大小：638K
- 年卷期：v.41,no.1(January 8, 2002)
- ISSN：1520-4995
文摘
The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichiacoli. The 
BORDER=0 > subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzymecomplex. consists of two domains: an N-terminal domain containing the proofreading exonucleaseactivity (residues 1-186) and a C-terminal domain required for binding to the polymerase (
) subunit(residues 187-243). Multidimensional NMR studies of 2H-, 13C-, and 15N-labeled N-terminal domains(186) were performed to assign the backbone resonances and measure HN-HN nuclear Overhauser effects(NOEs). NMR studies were also performed on triple-lableled [U-2H,13C,15N]186 containing Val, Leu,and Ile residues with protonated methyl groups, which allowed for the assignment of HN-CH3 and CH3-CH3 NOEs. Analysis of the 13C, 13C
beta2.gif" BORDER=0 ALIGN="middle">, and 13CO shifts, using chemical shift indexing and the TALOSprogram, allowed for the identification of regions of the secondary structure. HN-HN NOEs providedinformation on the assembly of the extended strands into a beta2.gif" BORDER=0 ALIGN="middle">-sheet structure and confirmed the assignmentof the helices. Measurement of HN-CH3 and CH3-CH3 NOEs confirmed the beta2.gif" BORDER=0 ALIGN="middle">-sheet structure andassisted in the positioning of the helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the activesite of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology.On the basis of this analogy, molecular modeling studies of 186 were performed using as templates thecrystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase andthe recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment wasconstructed, with the initial alignment taken from the previously published hidden Markov model andNMR constraints. Because several of the published structures included complexed ssDNA, we were alsoable to incorporate an A-C-G trinucleotide into the 186 structure. Nearly all of the residues whichhave been identified as mutators are located in the portion of the molecule which binds the DNA, withmost of these playing either a catalytic or structural role.
BORDER=0 > subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzymecomplex. consists of two domains: an N-terminal domain containing the proofreading exonucleaseactivity (residues 1-186) and a C-terminal domain required for binding to the polymerase () subunit(residues 187-243). Multidimensional NMR studies of 2H-, 13C-, and 15N-labeled N-terminal domains(186) were performed to assign the backbone resonances and measure HN-HN nuclear Overhauser effects(NOEs). NMR studies were also performed on triple-lableled [U-2H,13C,15N]186 containing Val, Leu,and Ile residues with protonated methyl groups, which allowed for the assignment of HN-CH3 and CH3-CH3 NOEs. Analysis of the 13C, 13Cbeta2.gif" BORDER=0 ALIGN="middle">, and 13CO shifts, using chemical shift indexing and the TALOSprogram, allowed for the identification of regions of the secondary structure. HN-HN NOEs providedinformation on the assembly of the extended strands into a beta2.gif" BORDER=0 ALIGN="middle">-sheet structure and confirmed the assignmentof the helices. Measurement of HN-CH3 and CH3-CH3 NOEs confirmed the beta2.gif" BORDER=0 ALIGN="middle">-sheet structure andassisted in the positioning of the helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the activesite of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology.On the basis of this analogy, molecular modeling studies of 186 were performed using as templates thecrystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase andthe recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment wasconstructed, with the initial alignment taken from the previously published hidden Markov model andNMR constraints. Because several of the published structures included complexed ssDNA, we were alsoable to incorporate an A-C-G trinucleotide into the 186 structure. Nearly all of the residues whichhave been identified as mutators are located in the portion of the molecule which binds the DNA, withmost of these playing either a catalytic or structural role.