Analysis of substrate specificity of the PaeR7 endonuclease: Effect of base methylation on the kinetics of cleavage

Ghosh, S. S., Obermiller, P. S., Kwoh, T. J., Gingeras, T. R. (1990) Analysis of substrate specificity of the PaeR7 endonuclease: Effect of base methylation on the kinetics of cleavage. Nucleic Acids Research, 18 (17). pp. 5063-5068. ISSN 03051048 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2402435
DOI: 10.1093/nar/18.17.5063

Abstract

In murine cells expressing the PaeR7 endonuclease and methylase genes, the recognition sites (CTCGAG) of these enzymes can be methylated at the adenine residue by the PaeR7 methylase and at the internal cytosine by the mouse DNA methyltransferase. Using nonadecameric duplex deoxyoligonucleotide substrates, the specificity of the PaeR7 endonuclease for unmethylated, hemi-methylated, and fully methylated N6-methyladenine (m6A) and C5-methylcytosine (m5C) versions of these substrates has been studied. The K(m), k(cat), and K(i) values for these model substrates have been measured and suggest that fully or hemi-m6A-methylated PaeR7 sites in the murine genome are completely protected. However, the reactivity of fully or hemi-m5C-methylated PaeR7 sites is depressed 2900- and 100-fold respectively, compared to unmodified PaeR7 sites. The implications of the kinetic constants of the PaeR7 endonuclease for these methylated recognition sites as they occur in murine cells expressing this endonuclease gene are discussed.

Item Type: Paper
Uncontrolled Keywords: dna methyltransferase endonuclease animal cell article DNA methylation enzyme kinetics enzyme specificity mouse nonhuman priority journal Adenine Animal Base Sequence Binding, Competitive Cytosine Deoxyribonucleases Type II Site-Specific DNA Modification Methylases Genes Hydrogen-Ion Concentration Kinetics Methylation Mice Molecular Sequence Data Oligodeoxyribonucleotides Substrate Specificity Temperature Animalia Felis catus Murinae
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation
organism description > animal > mammal > rodent > mouse
CSHL Authors:
Communities: CSHL labs > Gingeras lab
Depositing User: CSHL Librarian
Date Deposited: 13 Mar 2012 16:41
Last Modified: 28 Mar 2014 20:51
PMCID: PMC332124
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25246

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