From the PI-SceI is an intein-encoded protein
that belongs to the LAGLIDADG family of homing endonucleases. According
to the crystal structure and mutational studies, this endonuclease
consists of two domains, one responsible for protein splicing, the
other for DNA cleavage, and both presumably for DNA binding. To define
the DNA binding site of PI-SceI, photocross-linking was
used to identify amino acid residues in contact with DNA. Sixty-three
double-stranded oligodeoxynucleotides comprising the minimal
recognition sequence and containing single 5-iodopyrimidine
substitutions in almost all positions of the recognition sequence were
synthesized and irradiated in the presence of PI-SceI with
a helium/cadmium laser (325 nm). The best cross-linking yield
(approximately 30%) was obtained with an oligodeoxynucleotide with a
5-iododeoxyuridine at position +9 in the bottom strand. The subsequent
analysis showed that cross-linking had occurred with amino acid
His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A
variant of PI-SceI or in the presence of excess unmodified
oligodeoxynucleotide, no cross-linking was observed, indicating the
specificity of the cross-linking reaction. Chemical modification of His
residues in PI-SceI by diethylpyrocarbonate leads to a
substantial reduction in the binding and cleavage activity of
PI-SceI. This inactivation can be suppressed by substrate
binding. This result further supports the finding that at least one His
residue is in close contact to the DNA. Based on these and published
results, conclusions are drawn regarding the DNA binding site of
PI-SceI.
Homing endonucleases are a fascinating new class of enzymes that
cleave DNA with very high specificity within an extended recognition
site and, thereby, in vivo initiate a double strand break
repair that may lead to the insertion of the sequence coding for the
homing endonuclease into an allele that it lacks (for reviews, see
Refs. 1 and 2). They have been found in prokaryotes and eukaryotes as
well as in archaebacteria and are encoded by introns or inteins (for
review, see Ref. 3). The largest group is characterized by the presence
of one or two copies of a conserved dodecapeptide sequence, the
LAGLIDADG motif (4). PI-SceI, a homing endonuclease from
yeast, belongs to this group and occurs as an intein within the
vacuolar H+-ATPase, from which it is spliced in an
autocatalytic reaction (5).
The mature protein recognizes an extraordinarily long DNA sequence of
35-45 bp,1 bends the DNA,
and cleaves the substrate to produce a 4-bp 3' overhang (5, 6, 7). The
molecular details of DNA recognition and cleavage by PI-SceI
are unclear, in spite of the fact that the crystal structure of
PI-SceI is known (8). According to the structure analysis
and mutational studies (9), PI-SceI is composed of two
domains, one responsible for protein splicing (domain I) and one for
DNA cleavage (domain II), which are connected by two peptide segments.
The structure of the elongated domain I consists almost entirely of
All homing endonucleases have long recognition sequences (15-45 bp)
that can tolerate variation of the sequence, as shown for
PI-SceI (6) and other homing endonucleases, e.g.
I-CreI (17), I-DmoI (18), I-PorI (19),
I-PpoI (17), and I-TevI (20). Footprinting
studies with PI-SceI (6), I-DmoI (18), and
I-TevII (21) and their substrates show that they are
involved in both major and minor groove interactions. After cleavage,
PI-SceI (6, 7), I-SceI (22), F-SceII
(23), I-TevI (24), and I-TevII (21) remain bound
to one of the two cleavage products that may be required for the
subsequent recombination event which completes the homing reaction. The
genetically engineered domain DI of PI-SceI binds
specifically and with similar affinity as full-length
PI-SceI to DNA containing the PI-SceI recognition site as well as to one of the two cleavage products (9), demonstrating that domain I is not only involved in protein splicing but also in DNA
binding. In contrast, the genetically engineered domain II of
PI-SceI is not able to bind DNA with strong affinity,
suggesting that domain I is responsible for a decisive part of the
contacts between PI-SceI and its substrate (9). A similar
two-domain structure with a catalytic domain and a DNA binding domain
has been proposed for I-TevI (25).
The long recognition site of PI-SceI makes it an attractive
model for studying the mechanism of DNA sequence recognition by proteins but makes it difficult to model the DNA into the structure of
PI-SceI, in particular as it is known that both domains,
which may be linked in a flexible manner, are involved in DNA binding (9, 26, 27). In the study presented here, we tried to identify residues
of PI-SceI in close contact to the recognition site by a
photocross-linking technique using 5-iodine-substituted pyrimidines (5-IdU and 5-IdC). We show that PI-SceI can be cross-linked
via His-333 to a 5-Iododeoxyuridine (5-IdU) residue located in position +9 of the bottom strand, i.e. the right half of the
PI-SceI recognition sequence. Substitution of the
cross-linked amino acid His-333 by Ala results in a PI-SceI
mutant that is not significantly impaired in its ability to bind and
cleave DNA. However, no photocross-linking could be observed with the
H333A mutant, demonstrating that the region around His-333 is in close
contact with the DNA. With this information and knowing where the
active site is located, it is possible to present a model that
describes the approximate location of the DNA binding site in the
structure of PI-SceI.
Purification of Recombinant His6-tagged
PI-SceI--
His6-tagged PI-SceI was expressed
in Escherichia coli and purified as described by Wende
et al. (7).
Synthesis of Iododeoxyuridine- and Iododeoxycytidine-substituted
Oligodeoxynucleotides--
Oligodeoxynucleotides were chemically
synthesized by automated Photocross-linking of ds Oligodeoxynucleotides Carrying a Single
Iodopyrimidine Substitution and PI-SceI--
For analytical scale
photocross-linking, approximately 10 µM
PI-SceI was preincubated with 10 µM
radioactively labeled ds oligodeoxynucleotide monosubstituted with
5-IdU or 5-IdC at various positions of the top and bottom strand of
either the full-length recognition site or the right-half cleavage
product (cf. Fig. 1) in buffer P (10 mM
Tris/HCl, pH 8.5, 100 mM KCl, 2.5 mM EDTA) for
30 min at ambient temperature in a volume of 50 µl.
Photocross-linking was carried out with a 40 milliwatt helium/cadmium
laser emitting at 325 nm (Laser 2000). The total irradiation time was
usually 2 h; in kinetic experiments, 0-3 h. Samples of 2.5 µl
were withdrawn before and after cross-linking and analyzed on a 15%
(w/v) SDS-polyacrylamide gel. Gels were silver-stained and dried, and
radioactive bands were visualized by autoradiography with intensifying
screens or by using an imager. For preparative isolation of the
cross-linked PI-SceI/oligodeoxynucleotide complex (see
below), the analytical scale was increased 10-fold.
Photocross-linking of ds Oligodeoxynucleotide T + 9 and Nicked
PI-SceI--
PI-SceI (20 µM) was digested
under limiting conditions in 50 mM Tris/HCl, pH 8.0 with
trypsin at a substrate:protease ratio of 500:1 (w/w) at ambient
temperature, similar to that described recently (27). After 2 h of
incubation, the reaction was terminated by the addition of 5 mM phenylmethylsulfonyl fluoride. The cross-linking reaction with nicked PI-SceI (10 µM) was
performed in the presence of radioactively labeled ds
oligodeoxynucleotide T + 9 (10 µM, right-half cleavage
product) for 2 h in buffer P. 5-µl aliquots withdrawn before and
after irradiation were analyzed by electrophoresis on a 15% (w/v)
SDS-polyacrylamide gel with subsequent silver-staining and
autoradiography. Sequencing of the cross-linked C-terminal tryptic
fragment was performed as described recently (27).
Purification of the Cross-linked PI-SceI·Oligodeoxynucleotide T + 9 Complex--
The cross-linked
PI-SceI·oligodeoxynucleotide T + 9 (right-half cleavage
product) complex was purified from unreacted PI-SceI by
anion exchange chromatography on a Mono Q column (HR5/5, Amersham Pharmacia Biotech). After irradiation, the reaction mixture was incubated in the presence of 2 M urea for 5 min at 60 °C
and directly applied to the column. The elution buffers used were A, 50 mM Tris/HCl, pH 8.0 and B, 50 mM Tris/HCl, pH
8.0 with 1 M NaCl. The gradient applied was 0-80% B in 40 min. The flow-rate was 1.0 ml/min. The elution was monitored by
measuring the absorbance at 260 nm. Fractions of 1 ml were collected,
and aliquots were analyzed by electrophoresis on a 15% (w/v)
SDS-polyacrylamide gel.
Protease Digestions of the Cross-linked
PI-SceI·Oligodeoxynucleotide T + 9 Complex--
The purified
cross-linked complex of PI-SceI with oligodeoxynucleotide T + 9 obtained by anion-exchange chromatography was concentrated and
washed in a Centricon 50 tube with 50 mM Tris/HCl, pH 8.0. An aliquot (5 pmol) was radioactively labeled with
[ Identification of the Cross-link Site in the Cross-linked
PI-SceI·Oligodeoxynucleotide T + 9 Complex--
5 nmol of
PI-SceI were cross-linked with an equimolar amount of ds
oligodeoxynucleotide T + 9 (right-half cleavage product) by irradiation
for 2 h at 325 nm. The DNA in the reaction mixture was
subsequently radioactively labeled with [ Site-directed Mutagenesis, Purification, and Characterization of
the H333A Variant of PI-SceI--
Site-directed mutagenesis of the
PI-SceI gene was performed by a polymerase chain
reaction-based technique (29) using the primer
5'-GCTATGTTACTGATGAGGCCGGCATCAAAGCAACAATAAAG-3' to introduce the
desired mutation. The sequence of the mutated gene was confirmed by
sequencing. The purification of the PI-SceI mutant H333A was carried out as described for wild type PI-SceI (7). Binding and cleavage experiments were performed as described by Pingoud et al. (27), and bending assays were performed as described by Wende et al. (7). Photocross-linking experiments with the H333A variant and ds oligodeoxynucleotide T + 9 were carried out as
described above for wild type PI-SceI.
Generation of a 311-bp Recognition Site Containing a 5-IdU
Modification at Position +9 of the Bottom Strand--
A 311-bp
substrate carrying the PI-SceI cleavage site in the center
and the 5-IdU modification in position +9 of the lower strand was
generated by ligating two polymerase chain reaction products. The left
half, a 187-bp-long DNA fragment with the cleavage site and the
modification was produced with the primers
5'-GCGTCGGATCCAGGTCAAAGAGTTTTGG-3' and
5'-AGACTTCTGCCATTTCATTACCCTCXTTCTCCGCAC-3' (X,
5-IdU) and a 311-bp DNA fragment as template in the presence of
[ Modification of Histidine Residues in PI-SceI by
Diethylpyrocarbonate (DEPC)--
PI-SceI was dialyzed
against a buffer consisting of 30 mM inorganic sodium
phosphate, pH 6.4, 150 mM NaCl, and 20 mM
dithiothreitol. PI-SceI (6 µM) in the absence
and presence of equimolar amounts of a 62-bp substrate
(oligodeoxynucleotide G (7)) was treated with DEPC at final
concentrations of 0.25, 0.5, 1, 2.5, 5, and 10 mM for 30 min at ambient temperature. To remove the ethoxyformyl residue from the
histidine residues, an aliquot of the DEPC-treated mixture was
acidified by NaH2PO4 to reach a pH value of
6.25. Hydroxylamine was added to a final concentration of 250 mM, and the reaction mixture was incubated for 16 h at
4 °C. Circular dichroism spectra of 17 µM
PI-SceI before and after incubation with DEPC (3 mM) were measured in a buffer consisting of 30 mM inorganic sodium phosphate, pH 6.4, and 150 mM NaCl on a JASCO J-710 spectrophotometer at ambient temperature.
Electrophoretic Mobility Shift Assay--
For electrophoretic
mobility shift assays, PI-SceI was mixed with 10,000 cpm of
32P-labeled 311-mer polymerase chain reaction product
containing the PI-SceI recognition sequence in a total
volume of 10 µl of binding buffer (10 mM Tris/HCl, pH
7.5, 50 mM NaCl, 1 mM EDTA, 0.05% (w/v) nonfat
dry milk, 5% (v/v) glycerol, 10 mM dithiothreitol, 0.1 µg of poly(dI-dC)) (27). After electrophoresis, the gels were dried,
and bands were visualized and quantified in an imager.
To obtain detailed topological information about specific contacts
between PI-SceI and its DNA substrate, we performed
photocross-linking experiments using either 5-IdU or 5-IdC
monosubstituted ds oligodeoxynucleotides comprising the recognition
sequence for PI-SceI (Fig. 1).
Photocross-linking of halogenated pyrimidines has been used
successfully to identify contacts in DNA- and RNA-protein complexes
(30-34). 5-IdU is an almost perfect analogue of thymidine (35) and
particularly useful for the study of DNA-binding proteins using
oligodeoxynucleotides with a T Analytical Photocross-linking of PI-SceI·DNA Complexes--
We
used synthetic oligodeoxynucleotides comprising either the minimal
full-length recognition sequence of 36 bp (Fig. 1) or containing the
right-half cleavage product of the PI-SceI recognition sequence required for specific binding by PI-SceI (7). These oligonucleotides were substituted with a single 5-IdU moiety that substituted for T, A, or G, or with 5-IdC, which substituted for C, as
shown in Fig. 1. It was important to ensure that the presence of such a
substitution in the PI-SceI recognition sequence does not
interfere with the binding to PI-SceI. We therefore compared the binding of PI-SceI to modified and unmodified DNA in gel
shift experiments and found that they are bound by PI-SceI
with the same apparent KD of 5-10 nM as
the wild type sequence (data not shown). For analytical cross-linking
experiments designed to find out which position produces the best
cross-link yield, PI-SceI was incubated with the different
mono-substituted ds oligodeoxynucleotides for 30 min at ambient
temperature at a PI-SceI to DNA ratio of 1:1. The
photocross-linking reactions were carrried out by irradiation at 325 nm
with a helium/cadmium laser for 2 h at ambient temperature. Among
the various positions modified by 5-IdU or 5-IdC in the recognition
site, only three positions gave rise to a substantial amount of
cross-linked PI-SceI (Fig. 1). Oligodeoxynucleotides with
thymine in position +9 of the bottom strand and, to a somewhat smaller
extent, guanine in position +4 and adenine in position +5 of the upper
strand when substituted by 5-IdU, were efficiently cross-linked to
PI-SceI. The absence of significant amounts of cross-linked
product with DNA substituted at the other positions as shown in Fig. 1
indicates that either a suitable acceptor amino acid is not available
at these positions in sufficient proximity or the stereochemical
requirements for cross-linking are not fulfilled. Only the cross-link
to position +9 was investigated further, because the yield was high
enough to ensure that after purification of the cross-linked complex
and its proteolytic degradation, sufficient amounts of a
peptide/oligonucleotide adduct would be available for peptide sequencing.
Substitution of 5-IdU in position +9 of the bottom strand results in an
apparently homogenous cross-linked species as judged by SDS-PAGE (Fig.
2). The time course of irradiation of the
PI-SceI·oligodeoxynucleotide T + 9 complex is shown on a
silver-stained gel (Fig. 2A) and on its autoradiogram (Fig.
2B). The yield of this photocross-linked complex was
routinely about 20-30% after 2 h of irradiation and could not be
further increased by longer irradiation. Omission of PI-SceI
or incubation without irradiation failed to yield any cross-linked
material, nor could unmodified DNA be cross-linked to
PI-SceI to a significant extent.
Analytical Photocross-linking of Nicked PI-SceI·DNA
Complexes--
To find out whether the cross-link position is
localized in the C- or N-terminal half of the endonuclease, we
performed photocross-linking with nicked PI-SceI that was
generated by limited tryptic digestion under native conditions (27),
with the ds oligodeoxynucleotide substituted by 5-IdU in position +9 of
the bottom strand. Trypsin cleaves PI-SceI preferentially
after Arg-277 between Preparative Cross-linking of PI-SceI·DNA Complexes, Isolation,
and Proteolytic Digestion of Cross-linked PI-SceI--
After
preparative cross-linking of PI-SceI and
oligodeoxynucleotide T + 9 (right-half cleavage product), the resulting
mixture was resolved using anion-exchange chromatography. The eluted
fractions were analyzed by SDS-PAGE, and the covalently-linked
protein·DNA complex was identified as the peak eluting at 500 mM NaCl immediately before the free DNA (Fig.
4). Peak fractions contained the
PI-SceI·oligodeoxynucleotide T + 9 complex with >95%
purity.
To find out which protease would be most suitable to obtain a small and
defined fragment of CL PI-SceI, extensive proteolytic digestions were performed with aliquots of the radioactively labeled cross-linked PI-SceI·oligodeoxynucleotide T + 9 complex in
the presence of increasing amounts of trypsin, chymotrypsin, proteinase K, and subtilisin at 37 °C overnight. The degree of digestion was
analyzed by polyacrylamide gel electrophoresis in the presence of urea
and visualized by autoradiography (Fig.
5). The treatment of the
photocross-linked PI-SceI·oligodeoxynucleotide T + 9 complex with specific and unspecific proteases converted most of the
cross-linked PI-SceI·DNA complex to small cross-linked
peptides. The chymotryptic degradation was chosen for further
analysis, because it produced an apparently defined end product that,
because of the specificity of chymotrypsin, is likely to be homogenous.
(We found out later that apparently homogenous end products obtained
with proteinase K upon sequencing turned out not to have a defined
N-terminal sequence).
Identification of the Cross-link Position in the Cross-linked
Chymotryptic Peptide--
To identify the amino acid residue of the
homing endonuclease PI-SceI attached to the 5-IdU residue in
position +9 of the bottom strand of ds oligodeoxynucleotide T + 9, a
preparative cross-linking experiment with equimolar amounts of
PI-SceI and ds oligodeoxynucleotide T + 9 was carried out.
After irradiation and radioactive labeling, the reaction mixture was
incubated with chymotrypsin without prior purification of cross-linked
PI-SceI. An aliquot of the digested material was
precipitated and analyzed by polyacrylamide gel electrophoresis in the
presence of urea to confirm complete proteolysis of the cross-linked
PI-SceI·oligodeoxynucleotide T + 9 complex. The
cross-linked peptide was separated from other peptides by
anion-exchange chromatography on a Mono Q column (Fig. 6). Aliquots of the fractions 28-35 were
precipitated and analyzed on a urea polyacrylamide gel (Fig. 6).
Fractions containing the cross-linked peptide (fractions 32-34) were
combined, concentrated, precipitated and loaded onto a preparative urea
polyacrylamide gel. The cross-linked peptide/oligodeoxynucleotide T + 9 adduct appeared as a homogenous radiolabeled species with higher
apparent molecular weight than the free oligodeoxynucleotide strands
(not shown). It was extracted from the gel, desalted and lyophilized. The recovery was 400 pmols. The identity of the photocross-linked peptide/oligodeoxynucleotide adduct was determined by peptide sequencing. Only 50 pmols of the cross-linked peptide were accessible to sequencing. This low recovery of the chymotryptic fragment in the
sequencing analysis may be explained by adsorptive losses, losses
during the solubilization and washing procedure and to a large extent
by peptide modification by urea that is present in the purification
procedure. The peptide sequence covalently linked to position +9 in the
lower strand of the PI-SceI recognition sequence,
VTDEXGIKA, corresponds to amino acid residues 329 to 337 in
the PI-SceI protein sequence. At the fifth position (denoted by X) of the cross-linked peptide, where His-333 was
expected, no standard amino acid was identified by sequencing,
indicating that the major site of photocross-linking is at residue
His-333. This cross-link site resides in domain II, following the
second LAGLIDADG motif, which is essential for catalysis.
Loss of Photocross-linking Activity in the H333A Mutant
Protein--
Based on the identification of His-333 as the amino acid
covalently attached to the recognition site, the H333A variant of PI-SceI was produced. Both the binding and cleavage
properties of the PI-SceI mutant were characterized and
compared with the wild type enzyme. The H333A protein was shown in gel
shift experiments to bind to a polymerase chain reaction-generated 311 bp substrate with the same affinity as wild type PI-SceI. In
these experiments, two complexes can be observed, an upper complex and
a lower complex with low and high mobility, respectively. The two
complexes differ by the degree of DNA bending as shown for wild type
PI-SceI before (6, 7). Compared with wild type
PI-SceI, the lower complex is slightly less populated with
the H333A mutant. Cleavage properties of the H333A, however, are the
same as for the wild type PI-SceI, as shown independently by
He et al. (26). As expected, H333A was inactive in producing
a cross-link with the T + 9-substituted oligodeoxynucleotide (Fig.
7), indicating that His-333 is indeed responsible for the cross-link to ds oligodeoxynucleotide T + 9.
Absence of Effects of Cross-linking on Formation of Upper and Lower
Complex--
To find out whether cross-linking of DNA and
PI-SceI interferes with the formation of the upper or lower
complex observed in gel electrophoretic shift experiments with
PI-SceI (6, 7), we generated a 311-bp substrate containing a
5-IdU substitution in position +9 of the bottom strand. This substrate
was subjected to a photocross-linking reaction with PI-SceI.
In an analytical gel shift experiment, it could be demonstrated that
cross-linking had occurred, because a band shift is observed even in
the presence of an excess of a specific oligodeoxynucleotide competitor
added to the reaction mixture before loading the gel (data not shown). Under these conditions, the radioactively labeled 311-bp substrate T + 9 in the PI-SceI·DNA complex that had not been irradiated is replaced by unlabeled oligodeoxynucleotide F (7) and therefore does
not show a band shift. In addition, a preparative gel shift experiment
was performed with PI-SceI before and after cross-linking. Both, upper and lower complexes were extracted from the gel and analyzed by SDS-PAGE. Cross-linked DNA was shown to be present in both
complexes after irradiation (data not shown). This result suggests that
His-333 is in close proximity to T + 9 in the bottom strand in both the
upper and the lower complex or that the cross-linked PI-SceI/311-bp T + 9 adduct is in equilibrium between the
two conformations, characteristic for the upper and the lower complex.
Modification of His Residues by Diethylpyrocarbonate--
To test
whether His residues of PI-SceI are involved in DNA binding
and cleavage, a group-specific chemical modification of PI-SceI was performed using DEPC. The ethoxyformylation was
carried out with an increasing amount of DEPC in a 2-80-fold excess
over His residues. As shown in Fig. 8,
the modification of His residues in the PI-SceI molecule has
a dramatic effect on DNA binding. A 20-fold molar excess of DEPC over
His residues completely abolishes DNA binding by the endonuclease. In
the presence of equimolar amounts of DNA containing the recognition
site, the modification of His residues is slowed down, indicating that
these amino acids are protected from modification by the DNA and,
therefore, might be involved in a specific protein-DNA interaction. The
ethoxyformylation of His residues is in part reversible by treatment
with hydroxylamine, as shown by restoration of the binding activity.
This means that the DEPC treatment does not lead to an unspecific
denaturation of the enzyme but is a specific effect of the
ethoxyformylation of His residues. Experiments in which the cleavage
activity of DEPC-modified and -demodified PI-SceI was
measured produced nearly the same results, indicating that an impaired
DNA binding correlates with a reduced DNA cleavage activity. The
cleavage activity is restored by hydroxylamine as well. In fact,
circular dichroism analysis demonstrates that in the presence of 3 mM DEPC (10-fold molar excess of DEPC over His residues),
the secondary structure composition of PI-SceI is not
significantly different from that in the absence of DEPC (Fig.
8B). This result suggests that at least one His residue must
be involved in DNA binding and cleavage or located at the protein-DNA
interface such that its chemical modification interferes with DNA
binding and cleavage.
PI-SceI recognizes an extremely long asymmetrical
sequence of more than 30 bp in length, as shown by a primer extension
analysis (5), footprinting studies (12), and cleavage assays with substrates of different length (7). Specific binding is associated with
strong bending into the major groove (6, 7). The center of bending is
located at position +7, i.e. 5 and 9 nucleotides downstream
of the sites of cleavage in the upper and lower strands, respectively
(6, 7). Despite the fact that the crystal structure of
PI-SceI is known (8), the size of the recognition site makes it difficult to imagine how the DNA is bound. The authors of the crystal structure analysis proposed a docking model based on several criteria.
(i) The scissile phosphodiester bonds must be close to the two Asp
residues of the putative active site in domain II, Asp-218 and
Asp-326.
(ii) The center of the cleavage site has to be positioned such that the
left and right parts are in contact with the two symmetry-related (iii) The DNA backbone must be close to the positively charged residues
following the LAGLIDADG helices, at the interface between domains I and
II and in the extended region of domain I.
(iv) The bend of the DNA induced by specific complex formation has to
be implemented into the docking model.
The published model fulfils these criteria. Its characteristic feature
is that the DNA follows the concave contour of the PI-SceI
structure. According to this model, domain II interacts with about 14 bp, covering about 8 bp upstream and 6 bp downstream of the cleavage
site. The additional 16 or more bp on the right side extend to domain
I. It is conceivable that alternative docking models might satisfy the
criteria given by Duan et al. (8), and it is clear that it
would be very useful to know points of contacts between the protein and
the DNA to test alternative models. Presumably, the most
straightforward approach to determine such points of contact is to
produce cross-links between PI-SceI and its substrate. We
have chosen to use 5-iodopyrimidine-substituted DNA for our
cross-linking study because 5-iodouracil and 5-iodocytosine are
excellent chromophores to achieve photocross-linking of nucleoprotein complexes in high yield (35, 38, 31). In these studies up to 95% of
the modified nucleic acid could be cross-linked. A recent example in
which photocross-linking of 5-IdU-substituted double-stranded DNA to
protein has been reported is for Thermus aquaticus MutS, which is cross-linked with a Phe residue close to the N terminus to a
5-IdU-substituted heteroduplex DNA (33).
To find out which positions when substituted by 5-iodopyrimidines in
the recognition sequence of PI-SceI give rise to cross-links in good yield, we have synthesized 63 oligodeoxynucleotides
monosubstituted with 5-IdU (for T, G, and A) and 5-IdC (for C) in
almost all positions of the recognition sequence. These modified
oligodeoxynucleotides were hybridized with their complementary
unmodified sequence, incubated with PI-SceI in the absence
of Mg2+ (which is not required for specific binding), and
irradiated. 12 of 32 positions tested in the upper strand and 3 of 31 positions tested in the lower strand proved to be reactive in
photocross-linking, albeit with different yields (Fig. 1). The reactive
positions thus identified must be in close contact to the protein in
the PI-SceI·DNA complex and in the vicinity of a reactive
amino acid residue; depending on the extent to which these conditions
are fulfilled, different cross-linking yields are obtained. Our
cross-linking results indicate that reactive positions are found
preferentially in the upper strand with a cluster around the cleavage
site that is in good agreement with the results of methylation
interference experiments (6). The most reactive positions in the upper
strand are G + 4 and A + 5, 2 and 3 bp downstream of the site of
cleavage. The guanine residue was shown to be protected by
PI-SceI in a methylation interference experiment (6). The
lower strand contains only few reactive positions. The most reactive
position of all positions tested in the lower and upper strand,
however, was found in the lower strand at T + 9, 11 bp downstream of
the cleavage site. This region contains a run of pyrimidine residues
and, thus, was not informative in methylation experiments; it is,
however, a region shown to be in close contact with the protein by
hydroxyl radical footprint (12) and ethylation interference experiments (6). In addition, photocross-linking experiments in which the phosphate
group linking C + 6/T + 7 and T + 7/T + 8, respectively, in the lower
strand was substituted by a phosphorothioate group and coupled with
p-azidophenacylbromide produced cross-links in good
yield,2 confirming that this
region is in close contact with the protein.
We have selected position T + 9 in the lower strand for the
identification of a point of contact between PI-SceI and its
DNA substrate, as this position proved to be the most reactive of all
those tested. Furthermore, it is located approximately one helix turn
away from the site of cleavage and, therefore, promised to give useful
information regarding the location and orientation of the DNA substrate
in that part of the DNA binding site, which is far away from the
putative catalytic centers around Asp-218 and Asp-326.
Cross-linking experiments with nicked PI-SceI (27) and an
oligodeoxynucleotide modified at position T + 9 demonstrated that cross-linking had occurred to the C-terminal half of PI-SceI
(residues 278-454). Peptide sequencing of an isolated peptide
oligodeoxynucleotide adduct from the chymotryptic digest of the
cross-linked PI-SceI·DNA complex showed that the
cross-link involves His-333. In the PI-SceI structure, this
residue, located between Institut für Biochemie,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets, whereas the compact domain II is an almost equal mixture of
-helices and
-strands. Domain II is built up from two
substructures that are related by local 2-fold symmetry about an axis
between the two LAGLIDADG sequences. The domain II of
PI-SceI is structurally very similar to the homodimeric homing endonuclease I-CreI, which contains one LAGLIDADG
motif per subunit and lacks the protein splicing domain. As shown by the crystal structure analysis (10), the LAGLIDADG motifs in I-CreI form part of the dimer interface while simultaneously
positioning one of the conserved Asp residues adjacent to the scissile
phosphates. These residues may function to coordinate Mg2+
and thereby help to attack the DNA; substitution of these residues abolishes the endonuclease activity of I-CreI (11). Mutation of the analogous residues Asp-218 and Asp-326 in PI-SceI
also destroys the nucleolytic activity of this enzyme (12), whereas substrate binding of the mutated PI-SceI is not affected,
suggesting that these Asp residues are involved in catalysis. Similar
results were obtained with I-SceII (13), I-DmoI
(14), I-CeuI (15), I-PorI (14), and
PI-TliI (16), all members of the LAGLIDADG family of homing endonucleases.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyanoethylphosphoramidite DNA synthesis
using 5-IdU-
-cyanoethylphosphoramidites (Glen Research) on a Cyclone
plus DNA synthesizer (Millipore) or obtained by INTERACTIVA. To reduce
possible deiodination of the 5-IdU and 5-IdC, the final deprotection
step was carried out at ambient temperature for 24 h, as suggested
by the manufacturer. We used either the minimal full-length recognition
sequence (cf. Fig. 1) or the right-half cleavage product
comprising the upper strand
5'-GGAGAAAGAGGTAATGAAATGGCAGAAGTCT-3' (31-mer) and the lower
strand 5'-GATCAGACTTCTGCCATTTCATTACCTCTTTCTCCGCAC-3' (39-mer). Some
oligodeoxynucleotides were labeled at their 5'-terminus using T4
polynucleotide kinase and [
-32P]ATP.
-32P]ATP in the presence of T4 polynucleotide kinase.
Free [
-32P]ATP was removed using a NAP5 column
(Amersham Pharmacia Biotech). The radioactively labeled
PI-SceI·oligodeoxynucleotide T + 9 complex was digested in
50 mM Tris/HCl, pH 8.0, 1 mM CaCl2
by various proteases in a reaction volume of 50 µl. Trypsin and
chymotrypsin were added to give a final concentration of 2, 5, 10, 20, 40, 80, and 200 µg/ml. Proteinase K and subtilisin were added to give a final concentration of 2 µg/ml and 20 µg/ml. The digestions were
performed for 16 h at 37 °C. The reactions were terminated by
precipitation with 0.1 volume of 1 M sodium acetate, pH
6.8, and 2 volumes of ethanol. The samples were dissolved in 20 µl of
6 M urea, 0.025% (w/v) bromphenol blue, and 0.025% xylene
cyanole and subjected to electrophoresis on a 12% (w/v) polyacrylamide gel containing 0.5 × TBE and 2 M urea after a pre-run
for 30 min with a cathode buffer containing 1 mM
thioglycolic acid. The gel was dried, and radioactive bands were
visualized by autoradiography.
-32P]ATP and
T4 polynucleotide kinase. The buffer was adjusted to 50 mM
Tris/HCl, pH 8.0, 1 mM CaCl2, and 40 µg/ml
chymotrypsin was added. The digestion was performed for 2 h at
37 °C. To test the progress of chymotryptic proteolysis, an aliquot
of the reaction mixture was precipitated with ethanol and analyzed by
electrophoresis on a 12% (w/v) polyacrylamide gel containing TBE
(0.5× concentration) and 2 M urea. After the digestion was
complete, the whole sample was ethanol-precipitated, redissolved in 50 µl of 50 mM Tris/HCl, pH 8.0 and 2 M urea,
and applied onto a Mono Q column. For elution, the following buffers
were used: Buffer A, 50 mM Tris/HCl, pH 8.0; buffer B, 50 mM Tris/HCl, pH 8.0, and 1 M NaCl. The gradient applied was 0-80% B in 40 min. The flow rate was 1 ml/min. Fractions of 1 ml were collected, ethanol-precipitated, and redissolved in 6 M urea, 0.025% (w/v) bromphenol blue, and 0.025% (w/v)
xylene cyanole. 50-µl aliquots of the fractions were analyzed by
electrophoresis on a 12% (w/v) polyacrylamide, 0.5 × TBE, 2 M urea gel. For further purification of the cross-linked
peptide/oligodeoxynucleotide adduct, the peak fractions from the Mono Q
column were combined and subjected to preparative electrophoresis under
the same conditions as used for the electrophoretic analysis.
Radioactive bands were visualized by an imager. The cross-linked
peptide/oligodeoxynucleotide adduct was extracted from the gel and
eluted into 0.5 ml of 10 mM ammonium bicarbonate, pH 8.8, by shaking for 2.5 h at 37 °C. This solution was lyophilized,
and the cross-linked peptide/oligodeoxynucleotide adduct was
redissolved in 50 µl of 10 mM ammonium bicarbonate, pH
8.8, was applied to a 4-ml column of Sephadex G25 (Amersham Pharmacia
Biotech), eluted with the same buffer, and lyophilized. The recovery
was 400 pmols. For sequencing, the cross-linked
peptide/oligodeoxynucleotide complex was solubilized with 200 µl of
H2O and centrifuged using a ProSorb cartridge (Applied
Biosystems). The membrane was cut out, washed with 5% (v/v) methanol
for 5 min, and dried. The peptide was sequenced on a pulsed liquid
phase sequenator Model 477A (Applied Biosystems) with a 120A on-line
high performance liquid chromatography system according to Thole
et al. (28). 50 pmols of the material were amenable to sequencing.
-32P]dATP (7). For the generation of the right half,
a 124-bp DNA fragment, we used the primers
5'-GATGGAATTCCCAGAGTTATATC-3' and
5'-GCGTCGGATCCAAGCTTCTCTGGCTGC-3' and the unmodified 311-bp substrate as template, again in the presence of
[
-32P]dATP. Both DNA fragments were annealed with the
311-bp DNA by incubation at 95 °C for 10 min and, after cooling to
37 °C, ligated with T4 DNA ligase (AGS). The 32P-labeled
ligation product was purified by electrophoresis on a 10% (w/v)
polyacrylamide gel. The 311-bp substrate modified in position T + 9 of
the recognition site was incubated with 50 nM
PI-SceI and irradiated for 1.5 h at 325 nm as described
above. Analytical gel shift experiments were performed in the absence and presence of the 56-bp competitor oligodeoxynucleotide F (7). Preparative gel shift experiments of the PI-SceI/311-bp T + 9 complex were carried out before and after photocross-linking. Bands
corresponding to the upper and lower complex were excised, eluted in 50 mM Tris/HCl, pH 8.0, precipitated, and analyzed on a 6%
(w/v) polyacrylamide gel containing 1% (w/v) SDS.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5-IdU substitution in defined
positions. It is a photoactivated zero-length cross-linker that has the
advantage that cross-linking to regions of a protein not involved in
DNA binding is minimized and that irradiation with long wavelength UV
light (325 nm) does not lead to the excitation of other nucleic acid
and protein chromophores. Mechanistic studies of the 5-IdU chromophore
relevant to its use in nucleoprotein photocross-linking have been
performed by Norris et al. (36). The site-specific 5-IdU- or
5-IdC-mediated photocross-linking method does not depend on information
regarding the structure of the protein or the structure of the
protein·DNA complex. Efficient cross-linking requires the close
proximity of the modified base and a reactive amino acid. Preferential
targets are Phe, Tyr, Trp, His, and Met residues (35, 37). In addition,
the cross-link yield depends on a suitable orientation of the reacting
groups. With aromatic amino acid side chains as acceptor, the yield of the cross-linking reaction is significantly enhanced when a
-
stacking interaction between the base analog and an aromatic amino acid
residue is possible (36).
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Fig. 1.
Oligodeoxynucleotides used in the
photocross-linking reactions. Double-stranded
oligodeoxynucleotides comprising either the full-length
PI-SceI recognition sequence (in capital letters)
or the right-half cleavage product were monosubstituted with 5-IdU in
the cases of T, A, or G or 5-IdC in the case of C. The positions
(numbered as proposed by Gimble and Wang (6)) that were
substituted are indicated by circles or arrows.
Circles indicate positions that did not give rise to a
photocross-link, and arrows denote photoreactive
substitutions that lead to a photocross-link product with varying
yields. A cross-link in very good yields is observed with an
oligodeoxynucleotide substituted in position T + 9 of the bottom
strand, and two cross-links in good yield are detected with
oligodeoxynucleotides modified in position G + 4 and A + 5 of the upper
strand, whereas low cross-link yields are found for several additional
positions.
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Fig. 2.
Time course of the photocross-linking
reaction with PI-SceI and the ds oligodeoxynucleotide
T + 9. The PI-SceI·oligodeoxynucleotide T + 9 complex
was formed by incubating 10 µM PI-SceI with 10 µM 32P-labeled oligodeoxynucleotide
containing the 5-IdU residue in position +9 of the bottom strand
(cf. Fig. 1) for 30 min at ambient temperature. The mixture
was irradiated with a 40-milliwatt helium/cadmium laser emitting at 325 nm for the indicated time periods. In A, the silver-stained
gel of the irradiated samples analyzed by 15% (w/v) SDS-PAGE is shown.
The positions of cross-linked PI-SceI (CL
PI-SceI) and uncross-linked PI-SceI are
indicated. A protein contaminant of approximately 25 kDa is present in
the PI-SceI preparation. S represents the
molecular mass standard. In B, the autoradiogram of the gel
is shown.
-strand 16 and
-helix 6, separating the two
conserved LAGLIDADG motifs. The two resulting halfs comprise residues
1-277 and 278-454, which remain associated under native conditions
but dissociate upon treatment with SDS. DNA binding and cleavage
experiments had shown that nicked PI-SceI binds to substrate
DNA with essentially the same affinity as intact PI-SceI but
is devoid of DNA cleavage activity, possibly because it is not capable
of inducing the same strong distortion in its substrate DNA as
uncleaved PI-SceI, i.e. a bend of 75° (27).
Photocross-linking of native and nicked PI-SceI is compared
in Fig. 3. As shown in lane 2,
cross-linked PI-SceI migrates on an SDS-polyacrylamide gel
corresponding to a molecular species that is by 20-kDa larger than
uncross-linked PI-SceI. If the complex between nicked
PI-SceI and ds oligodeoxynucleotide T + 9 is irradiated and
the reaction mixture analyzed by SDS-PAGE, a cross-linked protein
fragment that migrates as a 40-kDa species (lane 4) is
observed. Based on these results, this species can only be interpreted
as a covalent adduct between the oligodeoxynucleotide modified in
position +9 of the bottom strand and the 20-kDa C-terminal half of
PI-SceI. This was confirmed by sequencing; the analyzed sequence was NNLNTENPLWDAIVG, which corresponds to the N terminus of
the cross-linked C-terminal peptide comprising residues 278-454.
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Fig. 3.
Photocross-linking of nicked
PI-SceI and ds oligodeoxynucleotide T + 9. PI-SceI was digested with trypsin under limiting conditions,
incubated with the 32P-labeled ds oligodeoxynucleotide T + 9, and irradiated for 2 h as described for PI-SceI.
Aliquots before and after irradiation were analyzed by SDS-PAGE on a
15% (w/v) gel. A shows the silver-stained gel, and B shows
the corresponding autoradiogram. S is the molecular mass
standard, lanes 1 and 2 represent the product
mixture obtained with PI-SceI, and lanes 3 and
4 represent that with nicked PI-SceI before and
after irradiation, respectively. CL PI-SceI denotes the
cross-linked PI-SceI·DNA complex, and CL C-term
represents the C-terminal fragment comprising amino acids
278-454 cross-linked to oligodeoxynucleotide T + 9. The positions on
the gel representing PI-SceI and the N- and C-terminal
fragments are indicated.
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Fig. 4.
Purification of the cross-linked
PI-SceI·DNA complex by anion-exchange
chromatography. 5 nmol PI-SceI were irradiated in the
presence of ds oligodeoxynucleotide T + 9 (5 nmol, right-half cleavage
product). The reaction mixture was applied to a Mono Q column that was
eluted by an increasing NaCl gradient from 0 to 0.8 M.
Individual fractions of 1 ml were collected, and 2.5-µl aliquots were
analyzed on a 12% (w/v) SDS-polyacrylamide gel. Free, uncross-linked
PI-SceI elutes in fractions 4 and 5 as monitored at 280 nm
in a parallel chromatogram. Lane S shows size markers,
lane 1 shows the reaction mixture before, and lane
2 shows the reactions mixture after irradiation for 2 h at
325 nm. Lanes 3, 4, 5, and
6 show aliquots of the fractions 29,
30, 31, and 32 after chromatography.
The gel was silver-stained. The positions on the gel
corresponding to CL PI-SceI and PI-SceI are
indicated.
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Fig. 5.
Digestion of the purified photocross-linked
PI-SceI·DNA complex with various proteases. The
isolated PI-SceI·DNA complex was digested in a volume of
50 µl of buffer (50 mM Tris/HCl, pH 8.0, 1 mM
CaCl2) either without or with increasing amounts of the
indicated protease (trypsin, chymotrypsin, proteinase K (PK)
or subtilisin (Sub)) for 16 h at 37 °C. After
ethanol precipitation, the digests were analyzed by 2 M
urea, 12% polyacrylamide gel electrophoresis in 0.5 × TBE
followed by autoradiography. The lane designated ds oligo
shows the positions of the two strands comprising ds
oligodeoxynucleotide T + 9 (right-half cleavage product). The
radioactivity in the two strands is different because of the lower
efficiency of labeling of the recessed 5'-end of the upper strand. The
lower strand is involved in the cross-link, whereas the upper strand is
released upon addition of urea. Small amounts of the lower strand seen
on the gel may be a contaminant carried over from the purification of
CL PI-SceI. Fig. 4 shows that there is no base-line
separation of CL PI-SceI and free
oligodeoxynucleotide.
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Fig. 6.
Purification of the cross-linked complex
after digestion with chymotrypsin and isolation of the
PI-SceI peptide involved in the cross-link.
Anion-exchange chromatography on a Mono Q column was used to separate
the free peptides after chymotryptic digestion (eluting in fractions 4 and 5) from the cross-linked peptide and the coeluting free
oligodeoxynucleotide. A260 was monitored, and
1-ml fractions were collected (upper panel). The
peptide/oligodeoxynucleotide adduct was identified by screening
aliquots of the fractions by 2 M urea, 12% polyacrylamide
gel electrophoresis in 0.5 × TBE followed by autoradiography.
Lane L represents an aliquot of the sample before
anion-exchange chromatography, and lanes 1-8 represent
aliquots of fractions 28-35 after precipitation. The position of the
cross-linked peptide/oligodeoxynucleotide T + 9 adduct (CL peptide) is
shown (lower panel). It is present in fractions 32 to
34.
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Fig. 7.
Photocross-linking of H333A to ds
oligodeoxynucleotide T + 9. Wild type PI-SceI, 5 µM (lanes 1 and 2) or the H333A
mutant, 5 µM (lanes 3 and 4) were
incubated with 5 µM ds oligodeoxynucleotide (right-half
cleavage product) containing 5-IdU in position +9 of the bottom strand
for 30 min at ambient temperature. The mixtures were irradiated for
2 h. Aliquots before (lanes 1 and 3) and
after irradiation (lanes 2 and 4) were analyzed
by electrophoresis on a 12% (w/v) SDS-polyacrylamide gel that was
silver-stained. Lane S shows size markers.
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Fig. 8.
Binding activity of PI-SceI
after modification and demodification of His residues with DEPC;
correlation with secondary structure. A,
PI-SceI (6 µM) was treated with increasing
concentrations of DEPC (0-10 mM) in the absence ( ) or
presence (
) of equimolar amounts of a 62-mer ds oligodeoxynucleotide
containing the PI-SceI recognition sequence. An aliquot of
the modified PI-SceI was demodified by treatment with
hydroxylamine (
, sample modified in the absence of DNA;
, sample
modified in the presence of DNA). For a gel shift analysis of the
binding of the DEPC-modified and -demodified PI-SceI to a
311-bp DNA substrate, PI-SceI aliquots (50 nM)
were incubated with 5 nM 32P-labeled 311-bp
substrate in the presence of 10 µg/ml poly(dI-dC). Bound and unbound
species were separated by electrophoresis on a native 6% (w/v)
polyacrylamide gel and quantified by an instant imager. B
shows the circular dichroism spectra of 17 µM
PI-SceI before (black curve) and after
modification by a 10-fold excess of DEPC over His-residues (gray
curve).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets 7 and 9, respectively, of domain II.
-strands 19 and 20, protrudes from the
protein surface into the cleft, which is generated by the junction
between the domains I and II (Fig. 9).
The fact that the H333A mutant cannot be cross-linked with an
oligodeoxynucleotide substituted in position T + 9 by 5-IdU
demonstrates the specificity of the cross-link. Although His-333 is in
contact with the DNA, its contribution to DNA binding and cleavage is
not significant, as the H333A variant binds and cleaves DNA with
similar efficiency as the wild type enzyme (this paper and Ref. 26). On
the other hand, given the length of the recognition sequence, large
effects are not expected for the H333A substitution, which only removes one of many interactions and is not expected to create a steric problem. This may be the reason why modification of PI-SceI
with DEPC, which leads to the ethoxyformylation of His residues and introduces a bulky group at this position, produces an enzyme defective
in binding and cleavage. Whether this effect is solely because of
modification of His-333 is not clear, in particular as two other His
residues in domain II, His-343 and His-377, are important for DNA
cleavage (26). That His-333 is likely to be located at the protein-DNA
interface can be inferred from a comparison of other LAGLIDADG-type
homing endonucleases. Although this residue is not conserved (39),
amino acid residues in I-DmoI and I-PorI, located
in a similar position as His-333 in PI-SceI, i.e.
6 and 4-10 amino acid residues, respectively, after the LAGLIDADG
motif, are protected against proteolytic attack by DNA binding
(40).
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Fig. 9.
Hypothetical model for the interaction
between PI-SceI and its bent DNA substrate. Based
on the structure of PI-SceI (Brookhaven Protein Data Bank:
1VDE), a schematic model is presented for the PI-SceI·DNA
complex in which the DNA containing the recognition site is bent by
approximately 70° and positioned such that the cleavage site is
located next to the presumptive catalytic Asp residues 218 and 326 and
the photocross-link position, His-333, next to thymidine in position +9
of the lower strand (both indicated in red). The protein
splicing domain (domain I) and the endonucleolytic domain (domain II)
of PI-SceI are located at the bottom and top of the
structure, respectively (8).
Our results demonstrate that this His-333 is in touch with the DNA in the upper and lower complex. If the scenario is correct that PI-SceI binds DNA first to form a complex (lower complex) in which the DNA is bent by 45° and then undergoes a conformational change to give a catalytically competent complex (upper complex) in which the DNA is bent by 75° (7), then the implication is that the region around His-333 remains in contact with DNA during the conformational change or does not interfere with it.
Conclusions--
The main purpose of the cross-linking study
presented here has been to find out how the DNA might be located with
respect to the enzyme in the specific PI-SceI·DNA complex.
Given the structural similarity between PI-SceI (8) and
I-CreI (10), there is no reasonable doubt that the DNA with
its scissile phosphodiester bonds is close to the Asp residues at the
ends of the LAGLIDADG motifs. This is one region that interacts with
the DNA. Another region has now been defined by a zero-length
cross-link to be around His-333. If conformational changes are not
excluded, involving for example loops connecting -strands 21 and 22 as well as 15 and 16 and possibly also a subdomain movement to better
position
-sheet 7, then this means that the DNA will be located in a
groove defined by
-sheets 7 and 9 in domain II, will leave domain II at His-333, cross the interdomain cleft, and make contact with domain I
via
-sheet 6,
-helix 1, and interconnecting loops. In contrast to
the docking model proposed by Duan et al. (8) in which the
bent DNA follows the concave contour of PI-SceI, in our
model the DNA takes up a convex curvature (Fig. 9). The comprehensive
mutational analysis carried out by He et al. (26) to support
the docking model of Duan et al. (8) is also in good
agreement with our model as are the hydroxyl radical footprint and
ethylation interference experiments (6, 12). It must be pointed out
that our model resembles with respect to domain II the docking model
proposed for I-CreI (10), a homodimeric homing endonuclease
with one LAGLIDADG motif per subunit. This similarity suggests that
PI-SceI, a monomeric protein with a pseudosymmetrical catalytic domain containing two LAGLDADG motifs like I-CreI,
has two catalytic sites that cooperate in cleaving the two strands of
the duplex substrate in one binding event (7). In this respect, PI-SceI presumably also resembles I-PpoI, the
only homing endonuclease for which a co-crystal structure has been
solved and for which it is understood how the DNA is bound in the
enzyme-substrate and enzyme-product complex (41).
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ACKNOWLEDGEMENTS |
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We thank Ms. Anja Wahl for excellent technical assistance and Drs. H. Sklenar and M. Zacharias for supplying the computer program CURVES.
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FOOTNOTES |
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* This work has been supported by grants from the Deutsche Forschungsgemeinschaft (Pi 122/13-1), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, and the Fonds der Chemischen Industrie.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.
This paper is dedicated to Professor Dr. Günter Maass on the occasion of his 65th birthday.
§ To whom correspondence should be addressed: Justus-Liebig-Universität Giessen, Institut für Biochemie, FB 15, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Tel.: +49 641 99-35402; Fax: +49 641 99-35409; E-mail: vera.pingoud{at}chemie.bio.uni-giessen.de.
Recipient of a scholarship of the Graduiertenförderung
des Landes Hessen.
2 F. Christ, unpublished information.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); CL, cross-link; DEPC, diethylpyrocarbonate; ds, double-stranded; 5-IdU, 5-iododeoxyuridine; 5-IdC, 5-iododeoxycytidine; PAGE, polyacrylamide gel electrophoresis; TBE, Tris borate/EDTA.
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