HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 10, 8235-8242, March 8, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/10/8235    most recent M107286200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Beard, W. A. Articles by Wilson, S. H. Search for Related Content PubMed PubMed Citation Articles by Beard, W. A. Articles by Wilson, S. H.

Loss of DNA Polymerase  Stacking Interactions with Templating Purines, but Not Pyrimidines, Alters Catalytic Efficiency and Fidelity^*

William A. Beard, David D. Shock, Xiao-Ping Yang, Saundra F. DeLauder, and Samuel H. Wilson^§

From the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and  Department of Chemistry, North Carolina Central University, Durham, North Carolina 27707

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, -helix N of DNA polymerase  is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

This article has been cited by other articles:

				P. Lin, V. K. Batra, L. C. Pedersen, W. A. Beard, S. H. Wilson, and L. G. Pedersen Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase {beta} PNAS, April 15, 2008; 105(15): 5670 - 5674. [Abstract] [Full Text] [PDF]

				G. C. Lin, J. Jaeger, and J. B. Sweasy Loop II of DNA polymerase beta is important for polymerization activity and fidelity Nucleic Acids Res., May 14, 2007; 35(9): 2924 - 2935. [Abstract] [Full Text] [PDF]

				W. W. Duym, K. A. Fiala, N. Bhatt, and Z. Suo Kinetic Effect of a Downstream Strand and Its 5'-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase {lambda} J. Biol. Chem., November 24, 2006; 281(47): 35649 - 35655. [Abstract] [Full Text] [PDF]

				M. C. Foley, K. Arora, and T. Schlick Sequential Side-Chain Residue Motions Transform the Binary into the Ternary State of DNA Polymerase {lambda} Biophys. J., November 1, 2006; 91(9): 3182 - 3195. [Abstract] [Full Text] [PDF]

				M. A. Kalam, K. Haraguchi, S. Chandani, E. L. Loechler, M. Moriya, M. M. Greenberg, and A. K. Basu Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. Nucleic Acids Res., January 1, 2006; 34(8): 2305 - 2315. [Abstract] [Full Text] [PDF]

				W. A. Beard, D. D. Shock, and S. H. Wilson Influence of DNA Structure on DNA Polymerase {beta} Active Site Function: EXTENSION OF MUTAGENIC DNA INTERMEDIATES J. Biol. Chem., July 23, 2004; 279(30): 31921 - 31929. [Abstract] [Full Text] [PDF]

				L. Yang, W. A. Beard, S. H. Wilson, S. Broyde, and T. Schlick Highly Organized but Pliant Active Site of DNA Polymerase {beta}: Compensatory Mechanisms in Mutant Enzymes Revealed by Dynamics Simulations and Energy Analyses Biophys. J., June 1, 2004; 86(6): 3392 - 3408. [Abstract] [Full Text] [PDF]

				R. Prasad, K. Bebenek, E. Hou, D. D. Shock, W. A. Beard, R. Woodgate, T. A. Kunkel, and S. H. Wilson Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase {iota} by Controlled Proteolysis J. Biol. Chem., August 8, 2003; 278(32): 29649 - 29654. [Abstract] [Full Text] [PDF]

				S.-J. Kim, W. A. Beard, J. Harvey, D. D. Shock, J. R. Knutson, and S. H. Wilson Rapid Segmental and Subdomain Motions of DNA Polymerase beta J. Biol. Chem., February 7, 2003; 278(7): 5072 - 5081. [Abstract] [Full Text] [PDF]

				W. A. Beard, D. D. Shock, B. J. Vande Berg, and S. H. Wilson Efficiency of Correct Nucleotide Insertion Governs DNA Polymerase Fidelity J. Biol. Chem., November 27, 2002; 277(49): 47393 - 47398. [Abstract] [Full Text] [PDF]

				E. Fidalgo da Silva, S. S. Mandal, and L. J. Reha-Krantz Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases J. Biol. Chem., October 18, 2002; 277(43): 40640 - 40649. [Abstract] [Full Text] [PDF]