Open AccessProbing DNA-protein interactionsin vitrowith the CpG DNA methyltransferase
Author(s) -
Stefan Kochanek,
Doris Renz,
Walter Doerfler
Publication year - 1993
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/21.10.2339
Subject(s) - biology , dna , microbiology and biotechnology , dna clamp , dna methyltransferase , biochemistry , circular bacterial chromosome , in vitro recombination , dna polymerase ii , cpg site , hmg box , dna footprinting , dna methylation , methyltransferase , gene , base pair , dna binding protein , methylation , complementary dna , polymerase chain reaction , gene expression , molecular cloning , reverse transcriptase , transcription factor
A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.