Sequence-specific DNA binding by the MspI DNA methyltransferase

Dubey, A. K., Roberts, R. J. (1992) Sequence-specific DNA binding by the MspI DNA methyltransferase. Nucleic Acids Res, 20 (12). pp. 3167-73. ISSN 0305-1048 (Print)0305-1048 (Linking)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/1535704
DOI: 10.1093/nar/20.12.3167

Abstract

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.

Item Type: Paper
Uncontrolled Keywords: Bacteriophage lambda/genetics Base Sequence Binding Sites/genetics DNA-Cytosine Methylases/*metabolism Kinetics Methylation Molecular Sequence Data Oligodeoxyribonucleotides/*metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > DNA binding protein
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > methyltransferase
CSHL Authors:
Depositing User: Matt Covey
Date Deposited: 23 Sep 2015 16:05
Last Modified: 08 Nov 2017 16:16
PMCID: PMC312454
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31842

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