Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.
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.
Kochanek
S
S
Institute for Genetics, University of Cologne, Germany.
Renz
D
D
Doerfler
W
W
D000256:Adenoviridae / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D013149:Spiroplasma / Q000201:enzymology; D014157:Transcription Factors / Q000378:metabolism; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC
D004279:DNA, Viral; D009687:Nuclear Proteins; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D003850:Deoxyribonuclease I