DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction.
EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.
Koudan
Elizaveta V
EV
Department of Chemistry, Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Russia.
Subach
Oksana M
OM
Korshunova
Galina A
GA
Romanova
Elena A
EA
Eritja
Ramon
R
Gromova
Elizaveta S
ES
D001483:Base Sequence; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000528:radiation effects*; D003488:Cyanogen Bromide / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D015342:DNA Probes; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000378:metabolism*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000528:radiation effects*; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D010777:Photochemistry; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D019920:Photoaffinity Labels; D004247:DNA; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D003488:Cyanogen Bromide; D003857:Deoxyuridine