From the Laboratoire de Pharmacologie des Macromolécules Biologiques, Institut Gustave Roussy, Villejuif, Cedex, 94805 France
Received for publication, February 7, 2001
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ABSTRACT |
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Despite its small size (27.6 kDa), the
group I intron-encoded I-SceI endonuclease initiates intron
homing by recognizing and specifically cleaving a large intronless DNA
sequence. Here, we used gel shift assays and footprinting experiments
to analyze the interaction between I-SceI and its
target. I-SceI was found to bind to its substrate
in monomeric form. Footprinting using DNase I, hydroxyl radical,
phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and
base-modifying reagents revealed the asymmetric nature of the
interaction and provided a first glimpse into the architecture of the
complex. The protein interacts in the minor and major grooves and
distorts DNA at three distinct sites: one at the intron insertion site
and the other two, respectively, downstream ( I-SceI is a homing endonuclease encoded by the mobile
group I intron of the large rRNA gene of Saccharomyces
cerevisiae (1, 2). This family of enzymes mediates the propagation
of the intron by cutting intronless genes at the site of intron
insertion (reviewed in Ref. 3). Like restriction enzymes, homing
endonucleases cleave double-stranded DNA with high specificity in the
presence of divalent metal ions. However, they differ from restriction endonucleases in their recognition properties and structures, as well
as in their genomic location (4). In particular, whereas restriction
enzymes have short recognition sequences (3-8
bp),1 homing endonucleases,
despite their small size, recognize long DNA sequences (12-40 bp).
They have been classified into four families on the basis of both their
sequence motifs and DNA cleavage mechanism (3). The protein
I-SceI is a member of the largest class of homing enzymes
(more than 130 proteins), characterized by the presence of either one
or two conserved 12 amino acid residue sequence motifs (LAGLI-DADG
motifs). Most of these proteins, like I-SceI, carry the
motif in duplicate and are endonucleases. I-SceI has been
purified as a monomeric globular protein of 235 amino acids (5). Its
endonuclease activity requires Mg2+ or Mn2+ but
not Co2+, Ca2+, Cu2+, or
Zn2+ to cleave DNA within its recognition sequence and
leaves a 4-bp overhang presenting a 3'-hydroxyl terminus (5, 6). The
enzyme displays a low turnover, probably because of its strong affinity for one of the products of the cleavage reaction (7).
The interaction of homing endonucleases with their substrates
raises an interesting question common to all the gene-regulatory proteins, namely: how can a small protein specifically recognize and modify a long DNA sequence? Understanding the molecular basis of
such a mechanism is essential for elucidating many aspects of cellular
control and is a prerequisite in any rational drug design program. It
is clearly established that local and global DNA structural features
that are highly sequence-dependent, play a primary role in
the dynamics of protein-DNA recognition (8). Sequence recognition would
arise from the inherent sequence-dependent ability to adopt
the conformation required for protein binding in the transient,
biologically active complex. In an attempt to reveal intrinsic helical
properties of the I-SceI nucleic acid target, we used this
sequence as substrate in earlier studies analyzing the mechanisms of
DNA chemical reactions and photosensitization processes (9, 10, 11,
12). We found evidence that the conformation of the helix deviates from
the ideal B-form duplex along two segments of three and five base pairs
located at a distance of approximately one helical turn, respectively,
upstream and downtream from the site of junction of the two exons (9).
In the present study, we first performed DNase I footprinting and gel
retardation assays to identify the complex formed between I-SceI and its target in the absence of a divalent metal
ion. We then used chemical probing agents to characterize the
conformation of DNA in the complex. I-SceI protein-DNA
complex was thus submitted to the nucleolytic attack of the cuprous
complexes of 1,10-phenanthroline (OP2Cu+ and
Phe-OP2Cu+), which results from the
abstraction of C-1' hydrogen atom by a tetrahedral copper-oxo species
bound within the minor groove (13, 14, 15, 9) and to
UVA/4',5'-dihydro-7-methylpyrido [3,4-c]psoralen (DHMePyPs)
photosensitization, which requires prior intercalation of the
pyridopsoralen at selective 5'-TTA-3' sites (10). We also employed
chemical modification agents of base and sugar residues (for a review,
see Ref. 16), diethyl pyrocarbonate (DEPC), which carboxylates purines
at the N-7 atom, potassium permanganate (KMnO4), which
oxidizes pyrimidine residues at the C5=C6
double bond, dimethyl sulfate (DMS), which primarily methylates the N-7
of guanine residues and free hydroxyl radical, generated by Fe-EDTA
reduction of hydrogen peroxide, which abstracts C-4' hydrogen atoms
from deoxyriboses of the DNA backbone. In the scheme that arises from
present experiments, I-SceI appears to stabilize, in
monomeric form, a constrained helical structure in which the minor
groove is widened at the cleavage sites. The results are discussed in
relation to previous reports on other endonucleases of the same family.
I-SceI Protein and DNA Substrates
I-SceI was purchased from Roche Molecular
Biochemicals, aliquoted at 10 units/µl in phosphate buffer in the
presence of 200 µg/ml bovine serum albumin, and conserved in 50%
glycerol at The 98-bp EcoRI-HindIII DNA fragment including
the I-SceI recognition sequence (sequence shown in Fig.
1A) was excised from its pUC19 vector supplied by B. Dujon
(6) and was purified by electrophoresis on a 15% preparative native
polyacrylamide gel as described (50). Concentration was measured by UV
absorbance. The fragment was stored in 10 mM Tris, pH 7.5, 1 mM EDTA (TE). For only 5' end-labeling, pUC19 plasmid
vector was first digested with either the restriction enzyme
EcoRI or HindIII, dephosphorylated with calf
intestine alkaline phosphatase, 5' end-labeled with T4 polynucleotide
kinase in the presence of [ Synthetic oligonucleotides used to form the 54- and 37-bp fragments
(Fig. 1, B and C), were purchased from Genset
(France). Purification, labeling, and annealing were carried out as
previously described (12).
Gel Shift Analysis of I-SceI/DNA Interactions
The conditions were derived from those described previously (17,
18). I-SceI (10 Quantification of the Apparent Equilibrium Dissociation
Constant
The apparent equilibrium dissociation constant was derived from
the Scatchard plot of the binding data in which the ratio of bound to
free DNA concentration was plotted against bound DNA concentration
using the Kaleidograph (Synergy software). The reciprocal of the
negative slope of the linear plot gives the value of the apparent
Kd.
DNA-Protein Stoichiometry
The 5' end-labeled DNA fragment (Fig. 1B,
54bp) (8 × 10 Complex Probing Using DNA Cleavage Reagents
Uniquely 5' end-labeled DNA fragments (Fig. 1, A,
B, or C) were digested directly or after
incubation with the I-SceI protein as described above. In
each case, digestion was carried out under conditions such that the DNA
molecule was broken only once.
DNase I Footprinting--
DNase I footprinting was done
essentially as previously described (51). Digestion was carried out at
23 °C using DNase I at a final concentration of 0.025 µg/ml for 15 or 45 s, depending on whether the DNA was free or
I-SceI-bound.
Hydroxyl Radical Footprinting--
A stock solution of
iron(II)-EDTA was prepared immediately before use by mixing equal
volumes of freshly prepared 0.4 mM
(NH4)2Fe(SO4)2,6H2O and
0.8 mM EDTA. The footprinting reaction was initiated by
placing iron(II)-EDTA solution (3 µl), 0.6% hydrogen peroxide (3 µl), and 20 mM sodium ascorbate (3 µl) on the inner
wall of the 1.5-ml Eppendorf tube containing 21 µl of free or
I-SceI-bound DNA, allowing the reagents to mix and then
adding the cutting reagent to the sample solution. The reaction was
allowed to run for 30 s and quenched by adding 3 µl of 1 M thiourea.
Phenanthroline Copper Complex
Footprinting--
Orthophenanthroline-cuprous complex
(OP2Cu+ or 5 Phe
OP2Cu+) footprinting was carried out as
previously described (29, 30). One µl of a solution freshly prepared
by diluting an ethanolic solution of 1,10-phenanthroline (1 mM) and an aqueous cupric sulfate solution (0.23 mM) was added to 10 µl of the appropriate free or
I-SceI-bound DNA sample. Cleavage was initiated by the
addition of 1 µl of 58 mM MPA (final concentration, 5.8 mM), and the mixture was incubated at 23 °C for
either 30 s for OP2Cu+ or 2 min for 5 Phe
OP2Cu+. Cleavage was quenched by the addition
of 1 µl of 28 mM 2,9-dimethyl-orthophenanthroline (final
concentration, 2.8 mM).
Analysis of Cleaved Fragments--
Specific quenching of the
footprinting reagent was followed by addition of a general stop
solution to a final concentration of 1 mM EDTA, 0.3 M sodium acetate, and 10 µg/ml tRNA. After phenol extraction, samples were ethanol-precipitated and lyophilized. The
dried samples were resuspended in 10 µl of gel loading buffer and
analyzed by denaturing gel electrophoresis in 15% (w/v) polyacrylamide containing 7 M urea. After electrophoresis, gels were dried
on Whatman 3MM paper and exposed to x-ray film (X-OMAT) for
documentation or to storage out of phosphor screens for quantification.
Complex Probing Using UVA DHMePyPs Photosensitization
UVA (365 nm) irradiation in the presence of DHMePyPs of the
I-SceI protein-bound DNA fragment (98 bp) was performed
exactly as previously described for free DNA (10). One µl of an
ethanolic psoralen solution (10 Complex Probing Using Nucleobase Modifications (52)
Whereas quite unreactive toward double-stranded adenine and
guanine residues, diethyl pyrocarbonate can carbethoxylate the N-7 atom
of purines of distorted structures, with a strong preference for
adenines, thus destabilizing the imidazole ring and creating a
piperidine-sensitive site. Similarly, because potassium permanganate oxidizes the C5=C6 double bond of pyrimidines (T Thymidines Using Potassium Permanganate
(KMnO4)--
One microliter of freshly prepared 0.1 M KMnO4 was added to 5 µl of the appropriate
labeled DNA sample. The reaction was stopped after 4 min at 23 °C by
addition of 2 µl of Adenines Using Diethyl Pyrocarbonate (DEPC)--
One microliter
of freshly prepared 3% DEPC was added to 10 µl of the appropriate
labeled DNA sample. The reaction mixture was incubated for various time
(30 s; 2.5 and 10 min.) at 30 °C and then stopped by addition of
10 µl of 50 mM imidazole. After phenol extraction,
samples were ethanol-precipitated, washed, and dried.
Guanines Using Dimethylsulfate--
The DNA fragment (Fig. 1),
uniquely 5' 32P-end-labeled on the top or on the bottom
strand, was methylated either directly or after incubation with
I-SceI protein (see above), by adding dimethyl sulfate
directly to the reaction mixture. The concentration of methylating
agent, reaction temperature, and incubation time were determined so as
to obtain in each case 1 N-7-MeG lesion per strand.
Processing of Modified DNA--
The pellets of modified DNA
samples were resuspended in 100 µl of 1 M piperidine at
95 °C for 30 min. Piperidine was removed by extensive
lyophilization. The dried samples were resuspended in 10 µl of gel
loading buffer (as described in ref. 50) and analyzed by denaturing gel
electrophoresis in 15% (w/v) polyacrylamide containing 7 M
urea. After electrophoresis, gels were dried on Whatman 3MM paper and
exposed to x-ray film (X-OMAT) for documentation or to storage out of
phosphor screens for quantification.
Quantification of Results
The autoradiograms were scanned by using a PhosphorImager and
Image Quant software (Molecular Dynamics). Measurements and normalization were carried out exactly as previously described (12).
I-SceI/DNA Binding; DNase I Footprinting, Affinity, and
Stoichiometry--
Analysis of the enzymatic activity of a collection
of mutations around the cleavage site has previously demonstrated that the minimal DNA recognition sequence for the I-SceI protein
extends over a continuous sequence of 18 base pairs, from positions I-SceI/DNA Minor Groove Interaction and DNA
Distortion--
Further analysis of the DNase I footprint by
converting band intensities to probabilities of cleavage at each site
relative to that in the control (see "Experimental Procedures")
shows that the degree of protection of discrete phosphodiester bonds
against the enzymatic digestion is not uniform. Areas from (
We then prepared a variant 98-bp DNA fragment (Fig. 1A) with
the substitution (G/C to A/T) at +7, which has been shown severely defective for I-SceI-mediated cleavage (6) and almost
completely resistant to I-SceI cleavage in
vitro(not shown). We incubated this fragment in the presence of
I-SceI and assayed either for binding in gel shift
experiments or for copper phenanthroline complex footprints.
No specific complex was observed in gel shift assays under the
conditions used previously with the wild-type DNA, but a smear appears
along the lane at high protein concentrations (Fig.
5). In the copper phenanthroline
footprints, we observe concomitantly the complete loss of the
downstream OP2Cu+ hole as well as that of the
5-PheOP2Cu+ hyperreactivity at +10 (not shown)
and the absence of the reactivity in the catalytic region (Fig. 7,
compare lanes 6 and 8; and not shown). Protein
binding in the downstream region thus appears directly related to the
induction of a conformational change in the catalytic region.
The Helix Is Sharply Kinked at the Junction between the Two
Exons--
To further investigate the structural change induced upon
protein binding at the junction of the two exons, we utilized a new UVA
triplet photosensitizer, 4',5'-dihydro-7-methylpyrido[3,4-c]psoralen (DHMePyPs), which induces thymine dimerization at selective sites in
DNA (10). The 98-bp DNA fragment (Fig. 1) 5' radiolabeled either at the
HindIII or EcoRI site, was exposed either
directly or after incubation with I-SceI protein, to UVA
irradiation (30 kJ m Evidence for Local Stacking Defects at Three Distinct
Sites--
Because local unstacking and/or unwinding of bases might be
expected as a result of helical distortion, we analyzed the enhancement of the sensitivity of adenine and thymine residues to diethyl pyrocarbonate and potassium permanganate, respectively (for a review,
see Ref. 16). DNA substrate, uncomplexed or complexed to
I-Sce1, was reacted with these reagents and cleaved at the modified bases by reaction with hot piperidine. The products were mapped at single-nucleotide resolution by analysis of the fragments produced on denaturing polyacrylamide gels. Fig.
7A (lanes 1-4) illustrates both the reactivity of double-stranded DNA with DEPC and
the influence of protein binding on this reaction. Cleavage observed at
guanines and to a much lesser extent at cytosines is caused by the
sensitivity of these residues to hot piperidine, totally independent of
DEPC treatment (control not shown). Only certain adenines of the top
strand display increased sensitivity to DEPC upon protein binding
(compare lanes 1 and 2). This enhancement is weak
at positions +1, +2, and +4, stronger at positions +9 and Methylation Protection and Interference--
Dimethyl sulfate
methylation has been used to probe DNA-protein contacts at the major
groove side of G/C base pairs (16). The methylation rate of the guanine
residues at positions I-SceI Protein-DNA Binding; Minor Groove Backbone Interactions and
DNA Distortion--
Specific contacts between the I-SceI
protein and the DNA backbone were monitored using free diffusible
hydroxyl radicals generated by hydrogen peroxide and iron(II) complexed
with EDTA (H2O2/Fe2+-EDTA) (35).
Fig. 9A shows representative
hydroxyl radical cleavage patterns of unbound and
I-SceI-bound DNA substrate. The alternation of
protected/unprotected areas can be observed on each strand. Optical
scans along typical lanes of the gel are given in Fig. 9B.
The data are schematized in Fig.
9C, where the major sites of
diminished and enhanced sensitivity are indicated. Protected sugar
residues appear in pairs lying across the minor groove from one another
and form three clusters separated by 4-5 base pairs where the cutting
efficiency of the backbone deoxyribose is much less affected by the
bound protein. In accord with DNase I results, these data thus identify
three consecutive loci on the minor groove, located on the same face of
the helix, where the protein contacts the DNA backbone. Consistent also
with the previous results, the upstream contact spot is very short and
the degree of protection weak compared to the catalytic and downstream
regions. Furthermore, comparison between lanes (1 to 4) and (5 to 8)
and also between lanes (9 to 12) and (13 to 16) shows that the protein
protects the bottom strand more than the top strand. In other respects, the binding of the protein results in the striking effect of rendering the substrate relatively more sensitive to hydroxyl radical attack at
the nucleotides adjacent to the cleavage sites C3 and
A4 on the top strand and T In the cellular context, the endonuclease I-SceI
discriminates its target site among ~107 bp (40). Present
results establish that, as with other proteins of the same family, the
absence of divalent metal ions eliminates cleavage but not
sequence-specific DNA binding. Consistent with the general tendency of
the two-motif LAGLIDADG homing endonucleases (4), I-SceI was
found to bind to its substrate in monomeric form. Furthermore, the
difference in the length of the downstream and upstream exonic
sequences involved in protein-DNA binding as well as the position of
the footprinting protection and cleavage maxima clearly reveal that the
downstream part of the recognition sequence is primarily involved in
I-SceI binding. Experimental results provide also the
evidence that I-SceI binding is accompanied by DNA
distortion. In the model of the complex that arises from the present
findings (summarized in Fig. 10 and 11), the binding of the protein
appears to distort its bound substrate to widen the minor groove at the
cleavage site and make the scissile phosphates accessible to the enzyme
active site. A sequential binding-kinking model is suggested in which
the first step of the protein binding would be facilitated by the
helical features of the sequence located at one helical turn downstream
from the intron insertion site. The high tendency of this region to
unwind is apparent from the OP2Cu+
hypersensitivity at positions 10 to 15 and from the strikingly high
sensitivity of position G7 to DMS methylation (Fig. 8),
observed to characterize guanines positioned in open DNA regions
(41). This intrinsic unwinding must facilitate protein
binding deep in the minor groove resulting in kinking the double helix
at the base step A9A10, 5' to the protein side
chain minor groove intercalation (42) and easily propagating helical
distortion. The induced helical distortion would position the
sugar-phosphate backbone of residues 2 and 5 on the top strand and The stabilization of a distorted DNA double helix appears to be a
common requirement for the homing endonucleases. From recent reports of
the high-resolution crystal structures of PI-SceI (44), I-DmoI (45), and I-CreI (46) and of the proteins
I-CreI or I-PpoI complexed to their DNA target
(47, 48), it appears that these relatively small homing endonucleases
utilize the same principle to recognize and cleave their long DNA
targets. They form extended folds that allow them to form long
interfaces across lengthy DNA homing sites and display preformed
binding motifs, consisting of antiparallel Chemical reactions sensitive to variations in helical conformation have
proved invaluable in revealing structural features of the free DNA
recognition sequence of the I-SceI protein. Here, using a
similar approach to study the DNA/I-SceI protein complex, we
established that the combination of such probes provides a valuable
means for extracting structural information about the structure of DNA
in the complex and relating it to intrinsic conformational features of
the double helix. The DNA sequence appears to show the protein the way
by which to position the catalytic machinery in proximity to the two
closely opposed scissile phosphates for nucleolytic attack. A
I-SceI/DNA cross-linking study is in progress to identify
protein-DNA contacts to further understand the molecular basis of such
a mechanism.
8,
9) and upstream
(+9, +10) from this site. The protein appears to stabilize the DNA
curved around it by bridging the minor groove on one face of the helix.
The scissile phosphates would lie on the outside of the bend, facing in
the same direction relative to the DNA helical axis, as expected for an
endonuclease that generates 3' overhangs. An internally
consistent model is proposed in which the protein would take advantage
of the concerted flexibility of the DNA sequence to induce a
synergistic binding/kinking process, resulting in the correct
positioning of the enzyme active site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Protein concentration was determined from the
optical density of the bands on a Phast System minigel (12.5%
acrylamide/5.5% SDS), using bovine serum albumin as internal standard
(not shown). The solution used in the present study had a concentration
of 0.72 × 10
12 mole per enzymatic unit.
-32P]ATP, and then digested
with the second restriction enzyme before purification by 20%
native polyacrylamide gel electrophoresis.
9 to 10
7
M) and 5' 32P-labeled DNA (either 98, 54, or 37 bp DNA fragments) (10
10 to 10
8
M) were preincubated separately for 2 min at 4 °C in the
binding buffer (final concentration: 10 mM Tris-HCl, pH 8, 10 mM NaCl, 2.5 mM dithiothreitol and 20 µg/ml bovine serum albumin) and then mixed and incubated for 10 min
at 20 °C. A 0.1 volume of loading buffer (50% glycerol/0.02%
xylene cyanol) was added. 8-12% polyacrylamide gels (29:1
acrylamide/bisacrylamide) were prerun in 1× TBE (9 mM Tris-HCl, 8.8 mM boric acid, and 2 mM EDTA) for 2 h at 120 V. Samples were loaded at 50 V, and the gels were run at 120 V at 4 °C. Gels were dried and
placed in phosphorimager cassettes. Screens were exposed for several
hours and scanned using a Molecular Dynamics PhosphorImager with Image
Quant software. The fraction of bound (free) DNA in each lane was
calculated by dividing the area of bound (free) bands by the total area
of bound and free bands. Each binding assay was performed in triplicate.
10 M) and
I-SceI protein (0.01 units/µl) were incubated as described above, before loading onto a set of 8, 10, 12, and 15% polyacrylamide gels, alongside 10 µg of nondenatured protein molecular size
standards (Sigma). Gels were stained with Coomassie Blue, destained,
dried, and exposed to x-ray film. The relative mobility of each species including free DNA fragment (Rf) was calculated by dividing the distance of the corresponding band by that of the bromphenol blue tracking dye in the same lane. For each species, the plot of 100[log (100Rf)] against gel concentration was constructed. The negative slope
or retardation coefficient (
Kr) was then
plotted as a function of the molecular mass for each protein standard,
and this calibration line was used to determine the apparent molecular
mass of the free DNA fragment and that of the protein
I-SceI/DNA complex. The difference between these two values
divided by the molecular mass of a protein monomer gives the number of
protein monomers bound to the DNA (n).
5 M) was
added to 19 µl of the appropriate labeled DNA sample in binding
buffer. After 10 min of incubation at room temperature in the dark,
sample-containing droplets were irradiated on ice at 365 nm using an
HPW 125 Philips mercury lamp at a fluence of 25 J/m2/s, as
determined by a VLX 365 radiometer. After irradiation, psoralen and
protein were extracted with chloroform/isoamyl alcohol/phenol followed
by G50-Sephadex column chromatography. The DNA was then ethanol-precipitated and treated as above.
C) from above or
below the plane of the base, these residues are susceptible to attack
by KMnO4 only if the stacking interaction is disrupted.
-mercaptoethanol. After phenol extraction,
samples were ethanol-precipitated, washed and dried.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 to +11 (Ref. 6; Fig. 1). However,
standard gel shift assays have so far remained unsuccessful (7), and
physical evidence of the in vitro binding of
I-SceI to its homing site has not been reported. To identify
the DNA sequence involved in the overall network of
interactions within the protein, we first subjected the
EcoRI-HindIII restricted DNA fragment from pUC18
(Fig. 1A, 98bp), containing the cloned junction between the
two exons of the intronless gene (6), to the limited nucleolytic
activity of DNase I before and after incubation with the I
Sce-I protein in the absence of Mg2+. It can be
observed (Fig. 2A) that the
binding of the protein not only protects DNA from
12 to +15 on the
top strand/
12 to +12 on the bottom strand but in addition induces
changes in the DNase I cleavage frequency from
20 to +28 (Fig.
2A). The 54-bp and 37-bp DNA fragments, containing or not
downstream distal sequences (Fig. 1, B and C)
were hence prepared and assayed for protein-binding in gel shift
experiments (17, 18). In both cases, we observed a discrete band of
decreased mobility (Fig. 3A
and not shown) reflecting the formation of a well defined complex. This
complex is specific because its formation could be competed by the
unlabeled DNA fragment but not by poly(dI-dC) (2 µg/ml) (data not
shown). Scatchard plots (19, 20, 21) of experiments varying either the
protein or DNA concentration yielded an apparent KD of 0.8 ± 0.03 nM and 8.4 ± 0.4 nM
when using, respectively, the 54-bp (Fig. 3B) or the 37-bp
(not shown) DNA fragments, thus showing that the interaction is
strengthened by the presence of distal downstream sequences. It is
interesting to note here that in experiments comparing the kinetics of
the initial phase of the cleavage reaction by incubating either of the
three DNA substrates (98, 54, and 37 bp) with an excess of protein in
the presence of 0.005 M MgCl2 (as previously
described in Ref. 7), the 37-bp DNA fragment was cleaved 1.4-1.6 times
more slowly than the 54-bp fragment, itself cleaved at a roughly
similar rate as the 98-bp DNA fragment (not shown). To examine the
possibility raised by these results that one protomer of
I-SceI (molecular mass, 27.6 kDa) binds to a single
DNA site, we then determined the molecular mass of the 54-bp
DNA/protein complex by using the Ferguson method (22, 23). In this
method, which requires that DNA fragments are not too long so that the
complex shape does not deviate significantly from globular (24, 25, 26,
27), the mobility of the complex is compared with that of standard
proteins in a set of nondenaturing gels of increasing polyacrylamide
concentration. Experiments using the 54-bp DNA fragment (Fig. 3,
C and D) lead to a molecular mass of 56.45 kDa
with a relative error estimated at ± 4.23 kDa over four trials.
Subtracting the contribution of free DNA yields an estimate of
26.71 ± 4.23 kDa for the molecular mass of the protein component
(Fig. 3E). The protein has appeared as a 26-kDa monomer in
solution (5).
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Fig. 1.
DNA sequences of the fragments used in this
study. The 18 bp DNA sequence, previously identified as the
minimal required for optimal cleavage activity, is marked by
brackets. IS and CS refer,
respectively, to the intron insertion site and the I-SceI
protein cleavage sites. Base pairs upstream and downstream from
IS are numbered from 1 and +1, respectively. A,
98-bp EcoRI-HindIII restriction DNA fragment from
pUC18 (6); B, 54-bp synthesized oligonucleotide duplex;
C, 37-bp synthesized oligonucleotide duplex.
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Fig. 2.
DNase I footprinting of
I-SceI on its DNA homing site. A, gel
analysis of the pattern of DNase I attack of the 98-bp
EcoRI-HindIII restriction fragment uniquely 5'
32P-labeled either on the top strand at the
HindIII site (lanes 1-4) or on the bottom strand
at the EcoRI site (lanes 5-8), naked
(lanes 2 and 6) or I-SceI
protein-bound (lanes 3 and 7). A+G Maxam-Gilbert
sequencing reactions were in lanes 1 and 4,
5 and 8, respectively. Numbers refer to the
position of the base with respect to the intron insertion site upstream
from ( 1) and downstream from (+1). B, schematic
representation of the influence of I-SceI binding on the
DNase I digestion frequency. The cleavage frequency is reduced by more
(open rectangles) or less (gray-filled
rectangles) than 70% and unchanged or enhanced
(black-filled rectangles). The bands at +13 on the top
strand and +1 on the bottom strand were taken as arbitrary references
of 100% inhibition.
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Fig. 3.
Binding of I-SceI to its DNA
substrate and stoichiometry. A, autoradiography of a
typical gel shift assay. A fixed concentration (10 9
M) of 5' 32P-labeled 54-bp DNA fragment was
incubated with increasing concentrations of I-SceI protein
in 10 mM Tris-HCl buffer, pH 7.9, containing 10 mM NaCl, 10 mM EDTA, 1 mM
dithiothreitol, and 20 µg/ml bovine serum albumin prior to loading on
a non-denaturing polyacrylamide gel (12%). From lanes 1-6,
I-SceI concentrations were: 0 M, 0.2 × 10
9 M, 0.7 × 10
9
M, 2 × 10
9 M, 4 × 10
9 M, 8 × 10
9
M. Protein-bound DNA migrates more slowly than free DNA
(indicated on the left). B, determination of the
apparent equilibrium dissociation constant (KD). Gel
shift assays using a fixed concentration of the I-SceI
protein and various concentrations of the 54-bp DNA fragment (Fig. 1)
were carried out as in A. The fraction of DNA bound to the
protein was calculated for each DNA concentration from the
radioactivity in the bands of the gels (see "Experimental
Procedures"). Scatchard analysis of the binding data (see
"Experimental Procedures") yielded a value of 0.8 ± 0.03 × 10
9 M for KD.
C, representative Ferguson analysis. A logarithmic function
of mobility for each of the protein standards and for free and
I-SceI-bound DNA (54 bp) was plotted against the
polyacrylamide concentration and fitted to a linear regression. Protein
standards were
-lactalbumin (a, 14.2 kDa), carbonic
anhydrase (b, 29.0 kDa), chicken egg albumin (c,
45.0 kDa); bovine serum albumin monomer (d, 66.0 kDa);
bovine serum albumin dimer (e, 132.0 kDa). The slope of each
line represents the retardation coefficient (Kr)
for each species. D, representative plot of
Kr versus molecular size
(MW). Kr values for protein standards
were plotted as a function of molecular size. Interpolation of
Kr values indicates a molecular size of 56.45 kDa for the I-SceI/DNA complex and 29.74 kDa for the free
DNA substrate. E, table of molecular sizes and
Kr values. To determine the value of the protein
component of the complex, we subtracted the molecular mass of the free
DNA contribution from that of the complex, giving 26.71 kDa for the
molecular mass of the protein in the complex.
12 to
2) on the top strand (
12 to
4) on the bottom strand and from (5 to 7) and (9 to 10) on the top strand and (3 to 6) on the bottom strand
are less protected than the intermediate segments (Fig. 2B).
Because DNase I hydrolyses the phosphodiester linkage of DNA from the
minor groove and because the geometry of B-DNA implies that the
positions closest to each other across the minor groove are 2-3 bp
apart in the sequence (28), the most protected regions identify the
minor groove of the catalytic domain and the next minor groove
downstream opening on the same face of the helix. In the next
experiment, the free and protein-bound 98-bp DNA fragment (Fig.
1A), 5'-radiolabeled at either the HindIII or
EcoR1 site, was subjected to nucleolytic attack by the
tetrahedral cuprous complex of 1,10-phenanthroline,
OP2Cu+ and its 5-phenyl derivative, 5-Phe
OP2Cu+. Fig.
4A shows the
OP2Cu+ and 5-Phe
OP2Cu+ cleavage patterns of DNA in the
absence and presence of I-SceI protein, whereas Fig.
4B gives the relative frequency of cleavage at individual
phosphodiester bonds (see "Experimental Procedures, Quantification of
Results"). Note first the significant variation of
OP2Cu+ cleavage frequency at individual
phosphodiester bonds along the DNA sequence in the absence of
I-SceI, reflecting the high binding specificity of the
tetrahedral coordination complex (Fig. 4; A, lanes
2 and 7; and B) (9). Binding of
I-SceI to DNA results, on the one hand, in the full
protection of the DNA segment delimited by the positions 7 and 13 on
the top strand 5 and 11 on the bottom strand and in contrast in the
increase in the OP2Cu+ cleavage frequency at
positions +2 to +4 on the top strand +1 to +3 on the bottom strand and,
though to a lesser extent, at positions
6 to
3 and
8 and
10 on
the top strand only. The positions more sensitive to
OP2Cu+ upon protein binding also exhibit
increased sensitivity to 5-PheOP2Cu+ but
interestingly, the phosphodiester bonds A9A10
on the top strand and A-7G-8,
G-8C-9 on the bottom strand are only
hyperreactive to 5-PheOP2Cu+ (Fig.
4A, compare lanes 3-4 and 8-9). The
inhibition of 1,10-phenanthroline copper-directed scission by a DNA
binding ligand may indicate either steric blockage of access to the
minor groove or/and a change of DNA geometry, resulting in poor
affinity for the tetrahedral coordination complex. Conversely,
hyperreactivity reveals protein-induced DNA deformation (29, 30, 31,
32). Our results thus identify three distinct distortion sites.
Furthermore, taking advantage of the complementarity of the two probes
OP2Cu+ and 5-PheOP2Cu+,
we can relate reactivity results to conformational features (see Fig.
4C). The presence of the bulky phenyl group, which decreases the overall kinetics of cleavage of free DNA by hindering entry of the
metallic complex inside the minor groove, probably helps stabilize the
metallic complex at a protein-induced helical distortion. 5-PheOP2Cu+, but not
OP2Cu+, has thus been shown to bind and
selectively cleave the sugar phosphodiester backbone of only one strand
at protein-induced alteration in the local DNA secondary structure and
thus detect subtle protein-induced DNA distortions (30, 31, 32). In the
downstream part of the recognition sequence, the striking association
of (i) the complete inability of OP2Cu+ to gain
access to the minor groove over roughly a half-turn of helix and (ii)
the top strand 5-PheOP2Cu+ hyperreactive site
right in the center of this area, suggests that DNA is distorted 5' to
minor groove protein I-SceI binding. Note that additional
evidence that minor groove protein binding deforms the helix is
provided by the DNase I sensitivity, in a stretch of protected
sugar-phosphodiester bonds, of the bond 9-10 on the top strand (Fig.
2, A and B; Ref. 28). Upstream, the increase in
OP2Cu+ reactivity at
6 to
3 on the top
strand only indicates that the minor groove can bind only one
phenanthroline ring close to the wall of the top strand. Together with
the hyperreactivity of 5-PheOP2Cu+ at the base
steps A
7G
8 and
G
8C
9 on the bottom strand, this result
indicates that the minor groove is either asymmetrically opened and/or
partially hindered by the protein (Fig. 4B). In the
catalytic region, the increased OP2Cu+
reactivity of the two strands and the reduction in the offset between
corresponding sugars (only 1 base pair) compared with that expected
with a normal B-form minor groove (2-3 base pairs) suggests that the
minor groove must be locally widened.
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Fig. 4.
OP2Cu+ and Phe
OP2Cu+ footprinting of I-SceI
on its DNA homing site. A, typical autoradiogram of a
12% sequencing gel showing the OP2Cu+
(lanes 2, 3 and 7, 8) and Phe
OP2Cu+(lanes 4, 5 and 9, 10) digestion pattern of the 98-bp DNA fragment, 5'
32P-end-labeled either on the top strand (lanes
1-5) or on the bottom strand (lanes 6-10), either
naked (lanes 2, 4, 7, and 9) or protein-bound
(lanes 3, 5, 8, and 10). A+G Maxam-Gilbert
sequencing reactions were in lanes 1 and 6.
Numbers refer to the position of the base with respect to the intron
insertion site, upstream from ( 1) and downstream from (+1).
Vertical bars show the location of
OP2Cu+ and Phe OP2Cu+
corresponding areas, and arrows indicate the sites of Phe
OP2Cu+ hyperreactivity. B,
quantitation of the relative probability of
OP2Cu+ cleavage at sites of each strand of DNA,
free in solution (stippled bars) or bound to
I-SceI (black bars). Peak heights, determined by
a PhosphorImager, are proportional to the probability of cleavage at
individual bases (see quantification in "Experimental Procedures").
Arrows indicate the sites hypersensitive to Phe
OP2Cu+ cleavage. C, schematic of
results. Filled and open horizontal
rectangles identify, respectively,
OP2Cu+ hyperreactive and protected areas.
Filled vertical rectangle crossing two filled
horizontal rectangles represent the two orthogonal phenanthroline
planes fitting the geometry of the minor groove from positions 1 to 4. One OP is deeply intercalated between base pairs, and the other OP is
close to the wall of the minor groove of one or the other strand (9).
Arrows indicate Phe OP2Cu+
hypersensitive sites.
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Fig. 5.
Comparison between
I-SceI binding to 98-bp wild-type and variant 98-bp
DNA substrate with the substitution (G/C to A/T) at +7.
I-SceI protein was incubated in 10 mM Tris-HCl
buffer, pH 7.9, containing 10 mM NaCl, 10 mM
EDTA, 1 mM dithiothreitol, and 20 µg/ml bovine serum
albumin with the 5' 32P-labeled 98-bp DNA fragment
substrate (10 9 M), either wild type
(lanes 1-2, 5-6, and 9-10) or
mutant (lanes 3-4, 7-8, and 11-12) prior to
either loading on an 8% non-denaturing polyacrylamide gel (lanes
1-4) or OP2Cu+ footprinting (lanes
5-12). Protein concentration was respectively 0.4. 10
9 M and 10
8 M in
the wild-type and mutant experiments. DNA fragments 5'
32P-end-labeled on the top strand or on the bottom strand
were, respectively, used in lanes 5-8 and
9-12.
2 at 365 nm) in the presence of
DH-MePyPs and then digested with the T4 DNA polymerase
3'-5' exonuclease activity (Fig.
6A). This enzymatic activity
is commonly used for quantitative detection of UV-induced DNA damage,
adducts, or pyrimidine cyclobutane dimers (33). The lesions
corresponding to blockage of the exonuclease activity at positions 1, +8, and +13 have been identified from T4 endonuclease-specific
targeting, in naked DNA, as the cyclobutane pyrimidine dimers
T1T2, T9T10 and
T14T15 (10). Free and protein-bound DNA
displayed significantly different cleavage patterns in the photosensitization experiment (Fig. 6A). The most striking
result is the dramatic increase (more than two orders of magnitude) in the frequency of termination of exonuclease activity at position
1.
This finding, reflecting the high frequency of dimerization of thymines
T1T2, indicates that binding of the protein
promotes intercalation of pyridopsoralen at the base step
T
1A1. Because the intercalation of the
pyridopsoralen proceeds via the minor groove (34), this reflects an
opening up of the minor groove, suggesting that I-SceI
binding at the site of junction of the two exons generates a kink
toward the major groove. Such a kink would induce the widening of the
flanking minor groove, in agreement with the
OP2Cu+ hyperreactivity at positions +2 to +4 on
the top strand and +1 to +3 on the bottom strand (Fig. 4, B
and C). A sharp change in the direction of the helix axis at
the step T
1A1 is also fully consistent with
the decrease in the OP2Cu+ reactivity of
positions +1, +2 on the top strand and positions
1,
2 on the bottom
strand in complexed compared to free DNA because the model of
OP2Cu+ binding involves the long axis of one
phenanthroline ring parallel with the minor groove axis (9). The
absence of the band corresponding to the dimer
T9T10 in the cleavage pattern of the bottom
strand agrees also with the protein binding-kinking proposed to
interpret copper complexes footprinting results (see above). Note also
the appearance of two new bands at +1 and +4 in that of the top strand, which have not, so far, be identified (Fig. 6).
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Fig. 6.
Influence of the binding of
I-SceI on the formation of photoproducts from DNA
pyridopsoralen photosensitization. A, comparison of the
T4 exonuclease digestion of the 98-bp DNA fragment, 5' 32P
uniquely end-labeled on the top strand (lanes 1 and
2) or on the bottom strand (lanes 3 and
4) after exposition to UVA (365 nm) in the presence of
DH-MePyPs, either naked (lane 1 and 3) or
protein-bound (lanes 2 and 4). Numbers refer to
the position of the base with respect to the intron insertion site,
upstream from ( 1) and downstream from (+1). B, schematic
representation of DH-MePyPs photosensitized I-SceI/DNA
complex damage. Arrows show the position of 3'-5'
exonuclease activity blockage, 3' to the damaged residue. Arrow
size correlates with the intensity of the bands. From the position
of these arrests on both strands, we deduce that there is a position
(filled vertical bar) where intercalation of pyridopsoralen
is highly favored when I-SceI is bound to its
substrate
6,
and stronger still at position
2 (Fig. 7B). In contrast, the adenines of the bottom strand are unreactive to DEPC, and the
residue at +8 even appears protected (compare lanes 3 and 4). The sensitivity of thymines to KMnO4 attack
is based on the comparison between guanine and thymine cleavage
frequencies. Cleavage does not occur at thymines in free DNA, whereas
in the protein-DNA complex, piperidine cleavage observed at some of the
thymines is similar to or greater than that observed at guanines. The
most dramatic increase in cleavage efficiency is observed at thymine T9 on the bottom strand and to a lesser extent at
T
1 on the top strand. The reactivity is also enhanced at
positions T
2, T+1, T+2,
T+4 on the bottom strand and T
7, T+8, T+11 on the top strand (Fig. 7;
A, lanes 5-8; and B). These
observations are in full agreement with the interpretation of
previous results.
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Fig. 7.
Influence of I-SceI
DNA binding on the modification of base residues. A,
DEPC modification of adenines and KMnO4 modification of
thymines. Typical autoradiogram of a sequencing gel showing the
modification of the DNA substrate, 5' 32P-end-labeled
uniquely on the top strand (lanes 1-2 and 5-6)
or on the bottom strand (lanes 3-4 and 7-8),
either free (lanes 1, 3, 5, and 7) or
protein-bound (lanes 2, 4, 6, and 8) using either
DEPC (lanes 1-4) or KMnO4 (lanes
5-8). Numbers refer to the position of the base with respect to
the intron insertion site, upstream from ( 1) and downstream from
(+1). The 54- and 98-bp DNA fragments were used in the DEPC and
KMnO4 experiments, respectively. B, diagram of
the reactivity of the I-SceI/DNA complex. The reactive
adenines and thymines are indicated by black-filled squares
(strongly reactive), gray-filled squares (moderately
reactive), and open squares (weakly reactive).
3,
4, and
5 and 5, 6, and 7 on the top
strand and 3 on the bottom strand is reduced in the complex compared
with free DNA (Fig. 8). We interpret this
result as indicating that the protein makes DNA contacts in the major
groove on either side of the cleavage sites but is not in intimate
contact with any of the guanine residues. In a second series of
experiments, the end-labeled DNA fragment was first methylated and then
incubated with the protein under similar conditions as those previously
defined when using non-modified DNA (see "Experimental
Procedures"). The bound fraction of modified DNA was then separated
by gel-shift from those species in which the modification prevents
protein binding. Isolated bound and unbound DNAs were then piperidine
cleaved at the modified positions and resulting fragments were analyzed
on denaturing polyacrylamide gels. Comparison of cleavage products of
DNA in the complex and in the remaining free DNA leads to the
conclusion that at guanine residues +5, +6, and +7, N-7 methylation
totally prevents the formation of the I-SceI/DNA complex
whereas it has only a slight effect at residues
3,
4, and
5 (Fig.
8A, lanes 3 and 4). This result again
underscores the primary importance of the downstream region.
Furthermore, because the methylation of guanine residues is merely
reduced but not totally inhibited, the suppression of an important
hydrogen bond between the protein and these residues does not appear to
be the cause for the strong interference of methylation of guanines +5,
+6, and +7 with protein binding. The data suggest that the inhibitory
effect of introducing a methyl group in the major groove of the
flanking region downstream of the cleavage site may be related, at
least in part, to hindering the establishment of the helical alteration
required for protein binding.
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Fig. 8.
DMS methylation and interference
results. A, 5' 32P-labeled DNA substrate
(54 bp) uniquely end-labeled on the top strand was subjected to DMS
methylation, either free (lane 1) or I-SceI-bound
(lane 2). 5' 32P-labeled DNA substrate uniquely
end-labeled on the top strand was DMS methylated prior to incubation in
the presence of I-SceI. Unbound DNA was separated from
I-SceI-bound DNA on a native polyacrylamide gel, cleaved
with piperidine, and analyzed on a denaturing gel. The
I-SceI-bound DNA fraction was loaded in lane 3 and unbound DNA in lane 4. B, extent of guanine
protection against DMS methylation by I-SceI DNA binding.
Peak heights are proportional to the probability of cleavage at
individual guanines of DNA, free in solution (stippled bars)
or bound to I-SceI (black bars). Black arrows
indicate the methylated guanines responsible for strong binding
interference
2 on the bottom
strand (Fig. 9B). One explanation might be that the reagent
is positioned in close proximity to the scissile bonds, resulting in a
local source of hydroxyl radicals. In this case, the stabilization
would imply that the reagent binds at the active site of the enzyme,
which normally chelates Mg2+. Indeed, it has recently been
reported that Fe2+ substituted at the active site of two
archaeal intron-encoded homing endonucleases yields functional enzymes
(36). Nevertheless, in our experiments, all the components of the
Fenton reaction had to be present for the effect to be observed (not
shown), making it unlikely that cleavage arises from the enzymatic
activity of the protein coordinated to free ferrous ions. In accord
with the alteration of the structure of the catalytic region upon
protein binding, the accessibility of the targeted bonds may rather
result from DNA bending in a direction, which compresses the major
groove and induces minor groove expansion (37, 38, 39). Note also, consistent with the local minor groove binding, the accessibility of the bonds 14-15 on the top strand and 10-11 on the bottom strand in a stretch of more or less protected sugar-phosphodiester bonds (Fig.
9, B and C).
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Fig. 9.
Hydroxyl radical footprinting of
I-SceI on its DNA homing site. A,
hydroxyl radical attack of the 54-bp DNA fragment 5'
32P-end-labeled uniquely on the top strand
(lanes 1-8) or on the bottom strand (lanes
9-16), either naked (lanes 1-4 and 9-12)
or protein-bound (lanes 5-8 and 13-16). Samples
were submitted to hydroxyl radical cleavage at 4 °C for 2 min
(lanes 1 and 5, 9 and 13) or 5 min
(lanes 2 and 6, 10 and 14) and at
37 °C for 1 min (lanes 3 and 7, 11 and
15) or 3 min (lanes 4 and 8, 12 and
16). Numbers refer to the position of the base with respect
to the intron insertion site, upstream from ( 1) and downstream from
(+1). Bands were assigned by reference to an A+G Maxam-Gilbert marker
track (not shown). B, densitometer scans of the hydroxyl
radical footprint along typical lanes of the autoradiogram. The
upper to lower tracings are respectively those of
lanes 2, 6, 10, and 14. C,
schematic representation of the relative intensity of individual maxima
in the presence or absence of the protein. Relative enhancement
of cleavage is indicated by arrows and protection by
open rectangles.
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Fig. 10.
Summary of the chemical probing data.
Upper, helical accessibility and protection from the inside
and the outside of the minor groove. Between the sequences of the two
DNA strands: filled and open horizontal
rectangles identify, respectively, OP2Cu+
hyperreactive and protected areas; the filled vertical
rectangle at the intron insertion site represents the intercalated
pyridopsoralen molecule. The filled vertical rectangle
crossing two filled horizontal rectangles represent the two
orthogonal phenanthroline planes (OP) fitting the geometry of the minor
groove from positions 1 to 4: one OP is deeply intercalated between
base pairs and the other OP is close to the wall of the minor groove of
one or the other strand (Schaeffer et al., Ref. 9).
Arrows indicate Phe OP2Cu+ hypersensitive
sites. On each side of the strands, the first lane indicates
hydroxyl radical protection (open rectangles) and
enhancement (arrows), and the second lane
represents the influence of protein binding on the DNase I cleavage
frequency (same symbols as in Fig. 2). Lower, base residue
modification and interference. The adenines and thymines reactive,
respectively, to DEPC and KMnO4 are indicated by
black-filled squares (strongly reactive), gray-filled
squares (moderately reactive), and open squares (weakly
reactive). The guanines protected against DMS methylation are indicated
by black-filled circles (strongly protected),
gray-filled circles (moderately protected), and open
circles (weakly protected). Black arrows indicate the
methylated guanines responsible for strong binding interference.
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Fig. 11.
Superimposition of the chemical probing data
on the helical representation of the I-SceI DNA homing site
(from 13 to +17). Blue ribbon areas indicate the
regions of the backbone (green ribbon) protected from
hydroxyl radical attack, and blue dashes indicate the minor
groove area protected from OP2Cu+ cleavage.
Red rectangles show the adenines and thymines that strongly
(filled rectangles), moderately (hatched
rectangles), or weakly (blank rectangles) react with
DEPC and KMnO4, respectively. Red dashes
indicate the binding domain of the tetrahedral coordination
complex OP2Cu+; red arrows show the
phosphodiester bonds hypersensitive to OP2Cu+
attack; and red asterisks the phosphodiester bonds
hypersensitive to hydroxyl radical attack. Black arrows show
the phosphodiester bonds hypersensitive to Phe
OP2Cu+ attack. The filled vertical
rectangle identifies the highly favored pyridopsoralen
intercalation base step. IS indicates the intron insertion
site. Bases are numbered from
1 and +1 extending, respectively,
upstream (on the left) and downstream (on the
right) from this site. The model was constructed using the
program Insight II (Molecular Simulations, version 98.0).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
to +3 on the bottom strand in register to be contacted by the protein
from the outside of the minor groove. This would result in the
induction of a new constraint that deforms the helical area
encompassing the cleavage sites. Note that the experiments using the
variant substrate with the substitution G/C to A/T at +7 support this
direct relationship. The protein would therefore be positioned closer
to the first steps of the next minor groove opening on the same side of
the helix, i.e. positions
5,
6 on the top strand and
7
on the bottom strand giving rise in turn to an upstream distortion
identified by the Phe-OPCu hypersensitivity at positions
8,
9, and
the unstacking of the bases A
6 and T
7.
Thus, by bridging the minor groove opening on the same face of the
helix, the protein would induce DNA to curve around it, the major
groove being directly accessible to the binding surfaces of the protein
on either side of the center of the homing site. The protein would thus
stabilize the natural tendency of the helix to bend, predicted by a
theoretical calculation using the program proposed by De Santis
et al. (43) according to which the DNA primary sequence
would induce a global curvature to the helix with a maximum distortion
angle at the step
A4G5.2
-ribbons, making extended
contacts with the DNA. However, the different conserved motifs that
characterize each family give each of them a specific interface
structure. In particular, the proteins of the LAGLIDADG family share a
domain fold characterized by the topology
. This
gives rise to an unusual
-ribbon helical interface whose
architecture displays an extensive curvature complementary to the DNA
major groove in the cleavage sites region and further facilitates the
recognition of extended DNA sequences (49). Nevertheless, the proteins
of the LAGLIDADG family differ greatly in the relative shapes and sizes
of their DNA binding surfaces (45).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank O. Mauffret and S. Fermandjian for access to their Molecular Simulations program and B. Dujon, H. Buc, and A. Travers for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant PO1009 from the Center National de la Recherche Scientifique and was performed in the Laboratoire de Physique et Chimie Biomoléculaires at Institut Curie and the Laboratoire de Chimie et Biochimie Pharmacologique et Toxicologiques at the Faculté de Médecine, Rue des Saint-Pères, Paris.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the French Ministère de
l'Enseignement Superieur et de la Recherche.
§ To whom correspondence should be addressed. Fax: 0147367470; E-mail: aspassky@igr.fr.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M101200200
2 F. Schaeffer, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bp, base pair(s); DEPC, diethyl pyrocarbonate; DMS, dimethyl sulfate.
![]() |
REFERENCES |
---|
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1. | Dujon, B., Gottarel, G., Colleaux, L., Betermier, M., Jacquier, A., d'Auriol, L., and Gallibert, F. (1985) in Achievement and Perspective of Mitochondrial Research II (Quagliariello, E. , Slater, E. C. , Palnieri, F. , Saccone, C. , and Kroon, A. M., eds) , pp. 215-225, Elsevier Sciences, Amsterdam |
2. | Colleaux, L., D'Auriol, L., Betermier, M., Cottarel, G., Jacquier, A., Galibert, F., and Dujon, B. (1986) Cell 44, 521-533[Medline] [Order article via Infotrieve] |
3. | Mueller, J. E., Bryk, M., Loizos, N., and Belfort, M. (1993) in Nucleases (Linn, S. M. , Lloyd, R. S. , and Roberts, R. J., eds), 2nd Ed. , pp. 111-143, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
4. |
Belfort, M.,
and Roberts, R.
(1997)
Nucleic Acids Res.
25,
3379-3388 |
5. | Montheilet, C., Perrin, A., Thierry, A., Colleaux, L., and Dujon, B. (1990) Nucleic Acids Res. 18, 1407-1413[Abstract] |
6. | Colleaux, L., D'Auriol, L., Galibert, F., and Dujon, B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6022-6026[Abstract] |
7. | Perrin, A., Buckle, M., and Dujon, B. (1993) EMBO J. 12, 2939-2947[Abstract] |
8. | Steitz, T. A. (1990) Quart. Rev. Biophys. 23, 205-280[Medline] [Order article via Infotrieve] |
9. | Schaeffer, F., Rimsky, S., and Spassky, A. (1996) J. Mol. Biol. 260, 523-539[CrossRef][Medline] [Order article via Infotrieve] |
10. | Andreu Guillo, L., Blais, J., Vigny, P., and Spassky, A. (1995) Photochem. Photobiol. 61, 331-335[Medline] [Order article via Infotrieve] |
11. | Andreu Guillo, L., Beylot, B., Vigny, P., and Spassky, A. (1996) Photochem. Photobiol. 64, 349-355[Medline] [Order article via Infotrieve] |
12. | Spassky, A., and Angelov, D. (1997) Biochemistry 36, 6571-6576[CrossRef][Medline] [Order article via Infotrieve] |
13. | Sigman, D. S., and Spassky, A. (1989) in Nucleic Acids Molecular Biology (Eckstein, F. , and Lilley, D. M. J., eds), Vol. 3 , pp. 13-27, Springer-Verlag, Berlin and Heidelberg |
14. | Sigman, D. S. (1990) Biochemistry 29, 9098-9105 |
15. | Yoon, C., Kuwabara, M. D., Spassky, A., and Sigman, D. S. (1990) Biochemistry 29, 2116-2121[Medline] [Order article via Infotrieve] |
16. | Nielsen, P. E. (1990) J. Mol. Recogn. 3, 1-25[Medline] [Order article via Infotrieve] |
17. | Fried, M., and Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505-6525[Abstract] |
18. | Garner, M. M., and Revzin, A. (1981) Nucleic Acids Res. 9, 3037-3060 |
19. | Sanders, C. M., and Maitland, N. J. (1994) Nucleic Acids Res. 22, 4890-4897[Abstract] |
20. | Withers, B. E., and Dunbar, J. C. (1995) Nucleic Acids Res. 23, 3571-3577[Abstract] |
21. | Wang, S., Cosstick, R., Gardner, J. F., and Gumport, R. I. (1995) Biochemistry 34, 13082-13090[Medline] [Order article via Infotrieve] |
22. | Ferguson, K. A. (1964) Metabolism 13, 985-1002 |
23. | Bryan, J. K. (1977) Anal. Biochem. 78, 513-519[Medline] [Order article via Infotrieve] |
24. | Bading, H. (1988) Nucleic Acids Res. 16, 5241-5248[Abstract] |
25. | Orchard, K., and May, G. E. (1993) Nucleic Acids Res. 21, 3335-3336[Medline] [Order article via Infotrieve] |
26. | Newman, M., Strzelecka, T. E., Dorner, L. F., Schildkraut, I., and Aggarwal, A. K. (1994) Nature 368, 660-664[CrossRef][Medline] [Order article via Infotrieve] |
27. | Mueller, J. E., Smith, D., Bryk, M., and Belfort, M. (1995) EMBO J. 22, 5724-5735 |
28. | Lahm, A., and Suck, D. (1991) J. Mol. Biol. 222, 645-667[Medline] [Order article via Infotrieve] |
29. | Spassky, A., and Sigman, D. S. (1985) Biochemistry 24, 8050-8056[Medline] [Order article via Infotrieve] |
30. | Thederahn, T., Kuwabara, M. D., Spassky, A., and Sigman, D. S. (1990) Biochem. Biophys. Res. Commun. 168, 756-762[Medline] [Order article via Infotrieve] |
31. | Frantz, B., and O'Halloran, T. V. (1990) Biochemistry 29, 4747-4751[Medline] [Order article via Infotrieve] |
32. | Spassky, A. (1992) Biochemistry 31, 10502-10509[Medline] [Order article via Infotrieve] |
33. | Doetsch, P. W., Chan, G. L., and Haseltine, W. A. (1985) Nucleic Acids Res. 13, 3285-3304[Abstract] |
34. | Demaret, J. P., Brunie, S., Ballini, J. P., and Vigny, P. (1989) Photochem. Photobiol. 50, 7-21[Medline] [Order article via Infotrieve] |
35. | Tullius, T. D., and Dombroski, B. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5489-5473[Abstract] |
36. |
Lykke-Anderson, J.,
Garrett, R. A.,
and Kjems, J.
(1997)
EMBO J.
16,
3272-3281 |
37. | Burkhoff, A. M., and Tullius, T. D. (1988) Nature 331, 455-456[CrossRef][Medline] [Order article via Infotrieve] |
38. | Yang, C. C., and Nash, H. A. (1989) Cell 57, 869-880[Medline] [Order article via Infotrieve] |
39. | Bennett, R. J., Dunderdale, H. J., and West, S. C. (1993) Cell 74, 1021-1030[Medline] [Order article via Infotrieve] |
40. | Thierry, A., Perrin, A., Boyer, J., Fairhead, C., Dujon, B., Frey, B., and Schmidt, G. (1991) Nucleic Acids Res. 19, 189-190[Medline] [Order article via Infotrieve] |
41. | Zaychikov, E., Denissova, L., and Heumann, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1739-1743[Abstract] |
42. | Werner, M. H., Gronenborn, A. M., and Clore, M. G. (1996) Science 271, 778-784[Abstract] |
43. | De Santis, P., Palleschi, A., Savino, M., and Scipion, A. (1990) Biochemistry 29, 9269-9273[Medline] [Order article via Infotrieve] |
44. | Duan, X., Gimble, F. S., and Quiocho, F. A. (1997) Cell 89, 555-564[Medline] [Order article via Infotrieve] |
45. | Silva, G. H., Dalgaard, J. Z., Belfort, M., and Van Roey, P. (1999) J. Mol. Biol. 286, 1123-1136[CrossRef][Medline] [Order article via Infotrieve] |
46. | Heath, P. J., Stephens, K. M., Monnat, R. J., and Stoddard, B. L. (1997) Nat. Struct. Biol. 4, 468-476[Medline] [Order article via Infotrieve] |
47. | Jurica, M., Monnat, R., and Stoddard, B. (1998) Mol. Cell. 2, 469-476[Medline] [Order article via Infotrieve] |
48. | Flick, K. E., Jurica, M. S., Monnat, R. J., and Stoddard, B. L.,. (1998) Nature 394, 96-101[CrossRef][Medline] [Order article via Infotrieve] |
49. | Philipps, S. E. V. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 671-701[CrossRef][Medline] [Order article via Infotrieve] |
50. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 1123-1128, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
51. | Spassky, A., Busby, S., and Buc, H. (1984) EMBO J. 3, 43-50[Abstract] |
52. | McCarthy, J. G., Williams, L. D., and Rich, A. (1990) Biochemistry 29, 6071-6081[Medline] [Order article via Infotrieve] |