Nucleic Acids Research, 2000, Vol. 28, No. 20 3962-3971
© 2000 Oxford University Press
Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase
Department of Biochemistry and College of Medicine, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA and 1Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
RsrI [N6-adenine] DNA methyltransferase (M·RsrI), which recognizes GAATTC and is a member of a restriction–modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M·RsrI were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M·RsrI binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M·RsrI bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M·RsrI extruding the target base from the duplex. Consistent with such base flipping, an 
1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M·RsrI to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M·RsrI–AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.
* To whom correspondence should be addressed. Tel: +1 217 333 2852; Fax: +1 217 333 6461; Email: gumport@uiuc.edu Present address: Sandra S. Szegedi, Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. A. Evdokimov, B. Sclavi, V. V. Zinoviev, E. G. Malygin, S. Hattman, and M. Buckle Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking J. Biol. Chem., September 7, 2007; 282(36): 26067 - 26076. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tamulaitis, M. Zaremba, R. H. Szczepanowski, M. Bochtler, and V. Siksnys Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence Nucleic Acids Res., July 9, 2007; 35(14): 4792 - 4799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Estabrook and N. Reich Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation J. Biol. Chem., December 1, 2006; 281(48): 37205 - 37214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Thomas and R. I. Gumport Dimerization of the bacterial RsrI N6-adenine DNA methyltransferase Nucleic Acids Res., February 6, 2006; 34(3): 806 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-J. Su, B. A. Connolly, C. Darlington, R. Mallin, and D. T. F. Dryden Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase Nucleic Acids Res., April 23, 2004; 32(7): 2223 - 2230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Thomas, R. D. Scavetta, R. I. Gumport, and M. E. A. Churchill Structures of Liganded and Unliganded RsrI N6-Adenine DNA Methyltransferase: A DISTINCT ORIENTATION FOR ACTIVE COFACTOR BINDING J. Biol. Chem., July 3, 2003; 278(28): 26094 - 26101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. V. Zinoviev, A. A. Evdokimov, E. G. Malygin, S. L. Schlagman, and S. Hattman Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. PROCESSIVITY AND ORIENTATION TO THE METHYLATION TARGET J. Biol. Chem., February 28, 2003; 278(10): 7829 - 7833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bheemanaik, S. Chandrashekaran, V. Nagaraja, and D. N. Rao Kinetic and Catalytic Properties of Dimeric KpnI DNA Methyltransferase J. Biol. Chem., February 28, 2003; 278(10): 7863 - 7874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Swaminathan, U. T. Sankpal, D. N. Rao, and A. Surolia Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase J. Biol. Chem., February 1, 2002; 277(6): 4042 - 4049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Cheng and R. J. Roberts AdoMet-dependent methylation, DNA methyltransferases and base flipping Nucleic Acids Res., September 15, 2001; 29(18): 3784 - 3795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. G. Malygin, A. A. Evdokimov, V. V. Zinoviev, L. G. Ovechkina, W. M. Lindstrom, N. O. Reich, S. L. Schlagman, and S. Hattman A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase Nucleic Acids Res., June 1, 2001; 29(11): 2361 - 2369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Scavetta, C. B. Thomas, M. A. Walsh, S. Szegedi, A. Joachimiak, R. I. Gumport, and M. E. A. Churchill Structure of RsrI methyltransferase, a member of the N6-adenine {beta} class of DNA methyltransferases Nucleic Acids Res., October 15, 2000; 28(20): 3950 - 3961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Szegedi and R. I. Gumport DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases Nucleic Acids Res., October 15, 2000; 28(20): 3972 - 3981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. K. Shier, C. J. Hancey, and S. J. Benkovic Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus J. Biol. Chem., April 27, 2001; 276(18): 14744 - 14751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vilkaitis, E. Merkiene, S. Serva, E. Weinhold, and S. Klimasauskas The Mechanism of DNA Cytosine-5 Methylation. KINETIC AND MUTATIONAL DISSECTION OF HhaI METHYLTRANSFERASE J. Biol. Chem., June 8, 2001; 276(24): 20924 - 20934. [Abstract] [Full Text] [PDF]
|
|