Abstract
The EcoRV methyltransferase modifies DNA by the introduction of a methyl group at the 6-NH2 position of the first deoxyadenosine in GATATC sequences. The enzyme forms a stable and specific complex with GATATC sequences in the presence of a nonreactive analogue, such as sinefungin, of its natural cofactor S-adenosyl-L-methionine. Using circular permutation band mobility shift analysis (in which the distance between the GATATC sequence and the end of the DNA is varied) of protein-DNA-cofactor complexes we have shown the methylase induces a bend of just over 60 degrees in the bound DNA. This was confirmed by phasing analysis, in which the spacing between the GATATC site and a poly(dA) tract is varied through a helical turn, which showed that the orientation of the induced curve was toward the major groove. There was no significant difference in the bend angle measured using unmethylated GATATC sequences and hemimethylated sequences which contain G6-Me ATATC in one strand only. These are the natural substates for the enzyme. The EcoRV endonuclease, a very well characterized protein, served as a positive control. DNA bending by this protein has been previously determined both by crystallographic and solution methods. The two proteins bend DNA toward the major groove but the bend angle produced by the methylase, slightly greater than 60 degree, is a little larger than that observed with the endonuclease, which is approximately 44 degrees.
Highlights
Vitro (Bracco et al, 1989)
The bend angles obtained using solution methods range from 30°, for Cro protein binding to OR1 operator, to 112° for the GalR protein binding to OI operator (Kim et al, 1989)
Sinefungin, an analogue of the cofactor Sadenosyl-L-methionine (AdoMet)1 increased binding by a factor
Summary
Materials—The purification of the EcoRV methyltransferase has been described previously (Nwosu et al, 1988; Szczelkun and Connolly, 1995). Construction of DNA Fragments for Bending Analysis—A set of six different 141-bp DNA fragments, with the EcoRV site “permutated” (i.e. placed at different distances from the end of the fragment) were constructed by PCR amplification of a pBend derivative (Zwieb and Adhya, 1994) which had an additional insert of 20 bp between the XbaI and SalI sites (Vipond and Halford, 1995) These plasmids were used as templates for PCR reactions with a common forward primer (CACTTTATGCTTCCGGCT) and five different reverse primers (phasing oligonucleotide 1, AGTCACGACGTTGTAAAAA; 2, TCACGACGTTGTAAAACG; 3, CGACGTTGTAAAACGACG; 4, ACGTTGTAAAACGACGGC; 5, GTTGTAAAACGACGGCCA) This produces five fragments of the same length (206 bp) with a GATATC EcoRV site phased with the poly(dA) tract (see Fig. 3). Determination of Bend Angles—The angle of curvature induced in the DNA by protein binding was calculated using the empirical equation of Thompson and Landy (1988); M/E ϭ cos(␣/2) (where M and E correspond to the electrophoretic mobility for the DNA-protein complexes when the binding site is at the middle and at the end of the DNA fragment, respectively)
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