Journal
ANALYTICAL BIOCHEMISTRY
Volume 381, Issue 1, Pages 135-141Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.06.035
Keywords
MBD; cleavage domain of endonuclease; CpG island; (5)mC-specific endonuclease; DNA methylation
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Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here. we describe the construction and characterization of two fusion endonucleases consisting of the (5)mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of Bmrl and Fold restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.Hpall (C(5)mCGG)-and M.Sssl ((5)mCpG)-modified DNA. Unmodified DNA and M.Mspl-modified DNA ((5)mCCGG) are Poor substrates for the C H-endonucleases. Sequencing cleavage products of modified lambda DNA indicates that cleavage takes place outside the (5)mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N(4-17) (5)mCpG, where / indicates the cleavage site). Such (5)mcpG-specific endonucleases will be useful to Study CpG island modification of the regulatory regions of tumor suppressor genes, and for the Construction of cell-specific and tumor-specific modified CpG island databases. (c) 2008 Elsevier Inc. All rights reserved.
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