Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships.
DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of approximately 40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.
Department of Molecular Medicine, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
Andrew R
AR
McNamara
Paul J
PJ
Hurd
Alexander E F
AE
Smith
Kevin G
KG
Ford
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D007700:Kinetics; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D013379:Substrate Specificity; D013997:Time Factors; D016335:Zinc Fingers / Q000235:genetics
D009841:Oligonucleotides; D004247:DNA; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases