LIGAND-INDUCED CONFORMATIONAL STATES OF THE CYTOSINE-SPECIFIC DNA METHYLTRANSFERASE M.HGAI-2

被引:19
作者
BALDWIN, GS
KELLY, SM
PRICE, NC
WILSON, GW
CONNOLLY, BA
ARTYMIUK, PJ
HORNBY, DP
机构
[1] UNIV STIRLING, DEPT BIOL & MOLEC SCI, STIRLING FK9 4LA, SCOTLAND
[2] NEW ENGLAND BIOLABS INC, BEVERLY, MA 01915 USA
[3] UNIV NEWCASTLE, DEPT BIOCHEM & GENET, NEWCASTLE UPON TYNE NE24HH, ENGLAND
关键词
M-HGAI; METHYLTRANSFERASE; S-ADENOSYL-L-METHIONINE; PYRIMIDINONE; 5-AZACYTOSINE;
D O I
10.1006/jmbi.1994.1012
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The interaction of one of the two DNA methyltransferases encoded by the HgaI restriction and modification system, M.HgaI-2, with substrates and substrate analogues is described. Circular dichroism spectroscopy has been used to demonstrate that addition of the methyl donor, S-adenosyl-L- methionine and the inhibitory substrate analogue sinefungin, both induce conformational transitions in the protein in the absence of DNA. Moreover, the addition of DNA is shown to enhance the apparent secondary structure of M.HgaI-2 whilst addition of sinefungin or S-adenosyl-L-methionine reduces apparent secondary structure. The circular dichroism spectrum of the abortive complex between the enzyme, DNA and sinefungin is dominated by the conformational properties of the binary complex of enzyme and sinefungin alone. Addition of a specific oligodeoxynucleotide duplex in which the target cytosine is replaced by a pyrimidinone, leads to a further ligand induced conformational transition as determined by electrophoretic analysis. The addition of sinefungin, or S-adenosyl-L-methionine, to M.HgaI-2 bound to the reactive oligodeoxynucleotide duplex, leads to yet another conformational transition in the protein as determined by the differential susceptibility of ternary and binary complexes to proteolysis. These experiments identify at least six ligand-inducible conformational states of M.HgaI-2 and, in view of the sequence similarity amongst this class of enzymes, suggest that conformational flexibility is a general feature of C-5 cytosine-specific DNA methyltransferases. Moreover, the substitution of the target cytosine by a pyrimidinone mimics the effect of 5-azacytosine incorporation into DNA.
引用
收藏
页码:545 / 553
页数:9
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