Footprint analysis of the
Bsp
RI DNA methyltransferase-DNA interaction
Footprint analysis of the Bsp RI DNA methyltransferase-DNA interaction
Csaba
Finta
and
Antal
Kiss*
Institute of Biochemistry, Biological Research Center of the Hungarian Academy
of Sciences, PO Box 521,
Szeged
6701,
Hungary
Received March 26, 1997;
Revised and Accepted May 21, 1997
DDBJ/EMBL/GenBank accession no. X15758
ABSTRACT
The interaction between the GGCC-specific
Bsp
RI DNA methyltransferase (M.
Bsp
RI) and substrate DNA was studied with footprinting techniques using a DNA
fragment that was unmodified on both strands. Footprinting with DNase I
revealed an
~
14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence.
Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove
interactions play little role in sequence-specific recognition by M.
Bsp
RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The
hydroxyl radical footprint of M.
Bsp
RI differs markedly from the the footprint reported for the
Hha
I and
Sss
I methyltransferases. The pattern of protection from dimethylsulfate and
hydroxyl radicals suggests that the interactions of M.
Bsp
RI with DNA are similar to those detected in the co-crystal structure of the
Hae
III methyltransferase.
INTRODUCTION
DNA (cytosine-5)-methyltransferases (C5-MTase) catalyze the transfer of a methyl group from
S
-adenosyl-L-methionine (AdoMet) to the C5 carbon of cytosine in specific sequences (
1
). C5-MTases share a common architecture. They contain five more and five less
conserved amino acid sequence motifs and a variable region. The latter exhibits
little sequence homology between C5-MTases that recognize different sequences (
1
). It has been shown that sequence specificity is determined by the variable
region (
2
,
3
). The principles of the catalytic mechanism of methyl transfer are well
established (
4
,
5
).
Most of our understanding of specific C5-MTase-DNA interactions is based upon X-ray structures of the complexes formed by the
Hha
I and the
Hae
III methyltransferases (M.
Hha
I and M.
Hae
III) with DNA (
6
-
9
). The co-crystal structures of the two enzymes are in many ways similar: both
methyltransferases consist of two domains, they use mainly their small domain
containing the variable region for sequence-specific binding, DNA recognition occurs predominantly in the major groove
and both enzymes employ base flipping to access the substrate cytosine (
6
-
9
). Comparison of the co-crystal structures also revealed some important differences. One of them was the extensive rearrangement
of bases in the recognition complex of M.
Hae
III. Another difference was that the residues used by M.
Hae
III to make base-specific contacts were different from those of M.
Hha
I (
7
). The latter observation was not surprising because there is very little
sequence homology between the variable regions of M.
Hha
I and M.
Hae
III (
7
). In contrast, the residues forming base-specific contacts in the M.
Hae
III complex are conserved in three other C5-MTases
(M.
Bsp
RI, M.
Bsu
RI and M.
Ngo
PII) that recognize the same sequence (GGCC) as M.
Hae
III (
7
,
10
). The difference in the co-crystal structures between M.
Hha
I and M.
Hae
III and the sequence conservation in the variable regions of M.
Hae
III, M.
Bsp
RI, M.
Bsu
RI and M.
Ngo
PII led Verdine and co-workers to propose that the recognition mechanism of the other three GGCC- specific C5-MTases may be similar to that of
M.
Hae
III (
7
).
Further information on the interaction between C5-MTases and DNA was provided by footprinting experiments (
11
,
12
). One of these studies (
12
) revealed that M.
Hha
I and M.
Sss
I, two C5-MTases that recognize the sequence GCGC and CG respectively, display similar specific and non-specific contacts with DNA when bound to their target sequences. A comparison of the footprint phenotype and the co-crystal structure of M.
Hha
I showed that although there were differences in the backbone contacts identified by the two approaches, the footprint phenotype of M.
Hha
I was largely consistent with the crystal structure (
12
). The similarity of the M.
Hha
I and M.
Sss
I footprint phenotypes suggested that the position of the enzyme with respect to
the recognition sequence and the contacts with the DNA backbone might be very
similar even among C5-MTases that have different specificity. On the other hand, the differences
between the co-crystal structures of M.
Hha
I and M.
Hae
III hinted at a diversity that is perhaps greater than could be expected for
this highly homologous class of enzymes. It awaits further studies involving
other C5-MTases to better assess the mechanisms by which C5-MTases interact with the substrate DNA.
Here we report footprinting analysis of the GGCC-specific C5-MTase M.
Bsp
RI. M.
Bsp
RI is part of the
Bsp
RI restriction-modification system of
Bacillus sphaericus
(
13
). Like M.
Hae
III, it methylates the inner cytosine of the recognition sequence. Beyond the
general goal of a detailed footprint analysis of a C5-MTase, our specific aim was to test the prediction (
7
) that monospecific C5-MTases that recognize the sequence GGCC interact with the substrate DNA in
a similar fashion.
MATERIALS AND METHODS
Enzymes and chemicals
M.
Bsp
RI was purified from an
Escherichia coli
strain carrying the
bspRIM
gene. Construction of the overproducer and enzyme purification will be described elsewhere. Restriction endonucleases
Bam
HI and
Nhe
I and
E.coli
DNA polymerase I large (Klenow) fragment were purchased from Fermentas. Bovine
pancreatic DNase I was from Sigma, dimethylsulfate (DMS) from EGA Chemie, 1,10-phenanthroline from Aldrich, poly(dI[middot]dC) from Pharmacia, deoxyadenosine 5'-[[alpha]-
32
P]triphosphate from Izotop Intezet Kft. All other chemicals were analytical
grade commercial products.
Radioactive labeling of DNA fragment
The 152 bp
Bam
HI-
Nhe
I fragment of plasmid pBR322 was used for footprinting experiments. To obtain a
fragment radioactively labeled at one end, pBR322 plasmid DNA was cleaved with either
Bam
HI or
Nhe
I, then the ends were labeled by a filling-in reaction using
E.coli
DNA polymerase I large fragment and deoxyadenosine 5'-[[alpha]-
32
P]triphosphate. The DNA was subsequently cut with the other restriction enzyme (
Nhe
I or
Bam
HI) and the 152 bp fragment was purified from agarose gel. The DNA strand corresponding to the
Nhe
I (5') ->
Bam
HI (3') orientation (
14
) will be referred to as the A and the complementary strand as the B strand.
Preparation of the enzyme-DNA complex
Binding reactions contained ~6 nM
Bam
HI-
Nhe
I fragment labeled with
32
P at either end (0.5-2 * 10
5
c.p.m.), 0-2 [mu]M M.
Bsp
RI, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 7 mM [beta]-mercaptoethanol, 4 [mu]M sinefungin and 8 ng/[mu]l poly(dI[middot]dC) in a total volume of 25 [mu]l. Reaction mixtures were incubated at
room temperature for 5 min.
DNase I protection
The reaction mixture was prepared by adding to the enzyme-DNA complex 5 [mu]l of a solution containing 15 mM CaCl
2
, 60 mM MgCl
2
and 5 ng/[mu]l DNase I (
15
). After incubation at room temperature for 1 min, the reaction was stopped by
extracting the sample with 50 [mu]l phenol/chloroform, then the DNA was precipitated twice with ethanol.
DMS protection
The reaction mixture was prepared by adding to the enzyme-DNA complex 1 [mu]l 10% DMS (
16
). After incubation at room temperature for 3 min, the reaction was terminated by adding 6 [mu]l of a solution containing 1.5 M sodium acetate, pH 7.0, 1 M [beta]-mercaptoethanol and 250 [mu]g/ml tRNA. The DNA was precipitated twice with ethanol, then
dissolved in 150 [mu]l 1 M piperidine. Cleavage at the methylated guanines was performed by
heating the samples to 90oC for 30 min. To remove the piperidine, the DNA was extracted twice with n-butanol (
17
) and then lyophilized.
Hydroxyl radical protection
The reaction mixture was prepared by adding to the enzyme-DNA complex 2 [mu]l 75 mM Fe(II)-EDTA, 2 [mu]l 150 mM ascorbic acid and 1 [mu]l 0.3% H
2
O
2
. These concentrations were somewhat different from the concentrations typically
used in hydroxyl radical footprinting experiments (
18
,
19
). A higher than usual concentration of Fe(II)-EDTA was used to compensate for the 0.33% glycerol, which was in the
footprinting reaction as a component of the enzyme storage buffer, and we found
that H
2
O
2
inhibited DNA binding at concentrations >0.01%. After incubation at room temperature for 3 min, the reaction was stopped and the DNA was
precipitated by adding 90 [mu]l of a solution containing 3% glycerol, 0.13 M sodium acetate, pH 7.0, and
92% ethanol. The precipitated DNA was dissolved, extracted with phenol, then
precipitated again with ethanol.
1,10-Phenanthroline-copper protection
The reaction mixture was prepared by adding to the enzyme-DNA complex 2.5 [mu]l of a solution containing 1 mM 1,10-phenanthroline/ 0.25 mM CuSO
4
and 2 [mu]l 150 mM ascorbic acid (
20
). Incubation and further processing was as described for the hydroxyl radical
protection.
Electrophoresis of DNA fragments
DNA fragments precipitated after the footprinting reactions were dissolved in 2 [mu]l loading buffer containing 98% formamide, 10 mM EDTA, pH 8.0, 0.025% xylene cyanol, 0.025% bromphenol blue and run in 10%
polyacrylamide gels containing 8 M urea. The gel was visualized, scanned and
quantified using a phosphorimager (Molecular Dynamics 445 SI). Bands were identified by alignment with co-migrating fragments from Maxam-Gilbert A + G reactions (
21
).
RESULTS
We found in previous experiments, using a gel retardation assay, that M.
Bsp
RI could form a specific recognition complex with DNA fragments containing a
Bsp
RI site (not shown). For footprinting experiments reported in this paper we used
the
Nhe
I-
Bam
HI fragment of plasmid pBR322. This fragment contains a single
Bsp
RI site (
14
). The fragment labeled with
32
P at either end was incubated with 0.2-2 [mu]M M.
Bsp
RI and was then used directly for footprinting reactions. We included the AdoMet analog sinefungin in the binding buffer because previous experiments showed that it increased
complex stability (unpublished observations).
DNase I protection
DNase I footprinting revealed a protected region that, in addition to the
recognition sequence, included several nucleotides in both directions. The
protected region was longer on the A strand than on the B strand (Fig.
1
). Another difference was that the protected stretch on the A strand was longer
in the 3' direction, whereas on the B strand it was longer in the 5' direction. On the A strand, 4-5 nt were protected on the 5'- and 8-9 nt on the 3'-side. On the B strand, 7
nt were protected on the 5'- and 4-5 nt on the 3'-side of the recognition sequence. In the
cleavage pattern of the A strand there was a very strong band corresponding to
the tenth base in the 5' direction. A less pronounced but still strong band could be seen in an
almost equivalent position (the ninth base on the 5'-side of the GGCC sequence) in the cutting pattern of the B strand (Fig.
1
). The strong bands were absent in the cleavage pattern of free DNA, suggesting
that a conformational change induced by M.
Bsp
RI led to preferential cleavage.
