|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 46, 30524-30529, November 13, 1998
From the Superposition of the PI-SceI and
I-CreI homing endonuclease three-dimensional x-ray
structures indicates general similarity between the I-CreI
homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed
to bind DNA. At the putative endonucleolytic active sites, the
superposition reveals that two lysine (Lys-301 and Lys-403 in
PI-SceI and Lys-98 and Lys-98' in I-CreI) and
two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI
and Asp-20 and Asp-20' in I-CreI) are related by 2-fold
symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the
PI-SceI reaction pathway was reported previously. Here, we
demonstrate the significance of the active-site symmetry by showing
that alanine substitution at Lys-403 reduces cleavage activity by
greater than 50-fold but has little effect on the DNA binding activity
of the mutant enzyme. Substitution of Lys-403 with arginine, which
maintains the positive charge, has only a modest effect on activity.
Interestingly, even though the Lys-301 and Lys-403 residues display
pseudosymmetry, PI-SceI mutant proteins with substitutions
at these positions have different behaviors. The presence of similar
basic and acidic residues in many LAGLIDADG homing endonucleases
suggests that these enzymes use a common reaction mechanism to cleave
double-stranded DNA.
Homing endonucleases are a group of enzymes that mediate DNA
rearrangement processes (for reviews see Refs. 1-3). The genes that
encode these proteins are frequently associated with inteins and Group
I introns, and homing endonucleases initiate the mobility of these
elements to loci that lack them by creating double strand breaks. The
LAGLIDADG subfamily of homing endonucleases, including the yeast
PI-SceI protein, typically contain two LAGLIDADG motifs (also termed EN1 and EN3 (4) or Blocks C and E (5, 6)) separated by
approximately 110 residues, but some LAGLIDADG enzymes encoded by Group
I introns, such as I-CreI, are significantly smaller and
contain a single motif.
The crystal structure of the PI-SceI intein was recently
determined, the first for a homing endonuclease and a protein generated by protein splicing (7). The structure shows that the endonucleolytic and protein splicing active sites are situated in separate domains. Residues within the two LAGLIDADG motifs in the endonuclease domain form two parallel PI-SceI forms two protein-DNA complexes with its asymmetric
31-base pair recognition sequence in gel mobility shift experiments, one in which contacts are made between the protein splicing domain and
a region of the substrate (region II) that is situated adjacent to the
cleavage site, and a second that includes these contacts as well as
additional interactions between the endonuclease domain and the
cleavage site itself (region I) (9-11). By contrast, I-CreI interacts with a shorter, pseudosymmetric 19-24-base pair sequence to
yield a single complex (12).
Here, we report that a structural comparison of the PI-SceI
and I-CreI proteins (7, 13) reveals a lysine residue at the PI-SceI active site, Lys-403, that is related by local
2-fold symmetry to Lys-301. Analysis of mutant PI-SceI
proteins with substitutions at Lys-403 demonstrates the importance of
this residue in the reaction pathway. This previously unrecognized
symmetry relationship in PI-SceI necessarily implies that
two identical pairs of residues (Asp-218 and Lys-301 and Asp-326 and
Lys-403) form either one or two active sites. Superposition of
I-CreI and PI-SceI indicates that each pair of
residues overlaps with a similar pair in each of the symmetry-related
monomers of I-CreI and suggests that the LAGLIDADG homing
endonucleases share a common active-site architecture and catalytic mechanism.
Materials--
All oligonucleotides were synthesized by Genosys
Biotechnologies, Inc. TALON metal affinity resin was obtained from
CLONTECH, and SP-Sepharose was obtained from
Amersham Pharmacia Biotech.
Mutagenesis of the PI-SceI Gene--
Plasmid pET
PI-SceI C-His contains a PI-SceI gene that
encodes a 479-amino acid PI-SceI derivative with a
polyhistidine C-terminal extension (9). The K403A and K403R
substitutions were introduced into the PI-SceI gene using
mutagenic oligonucleotide primers in a two-step overlapping polymerase
chain reaction amplification protocol (14). All mutations and inserted
sequences were confirmed by dideoxy sequencing.
Expression of PI-SceI Protein--
Wild-type PI-SceI
and the PI-SceI mutant derivatives were purified as
described previously (9) by Co2+-metal affinity and
SP-Sepharose ion-exchange chromatography to greater than 95% as judged
by SDS-polyacrylamide gel electrophoresis. Protein concentrations were
determined using an extinction coefficient of 5.03 × 104/M/cm (9).
Assay of PI-SceI DNA Binding--
A 219-base pair duplex DNA
probe that contains a single PI-SceI recognition site was
prepared for native gel mobility shift assays by polymerase chain
reaction and end-labeled with [32P]ATP (9). Purified
wild-type PI-SceI and the K301A, K301R, K403A, and K403R
variant proteins (0.7 nM) were used in gel mobility shift
experiments to measure DNA binding as described previously (9).
Protein-DNA complexes were separated from unbound DNA on a 7% native
polyacrylamide gel and were visualized by autoradiographic exposure of
the dried gel to film.
PI-SceI-mediated DNA Cleavage--
Rates of DNA cleavage were
measured under single-cycle conditions where enzyme was in excess over
substrate. A linear plasmid substrate (pBS-PISce36, 7 nM) containing a single PI-SceI recognition site
was incubated as described previously with either purified wild-type,
K403A, or K403R proteins (100 nM) for various lengths of
time at 37 °C (9). The amounts of undigested linear plasmid DNA and
of the two cleavage products were determined by scanning densitometry
(Molecular Dynamics), and cleavage rates were determined by
curve-fitting of the data using KaleidaGraph (Synergy Software).
The yeast PI-SceI and the Chlamydomonas
I-CreI homing endonucleases, in common with all
characterized LAGLIDADG endonucleases, require Mg2+
co-factor for DNA cleavage activity and cut DNA to yield a 4-base pair
extension with 5'-phosphate and 3'-hydroxyl groups (1), thus raising
the possibility that these enzymes utilize a common reaction mechanism.
An important functional difference between the proteins is that
PI-SceI, but not I-CreI, catalyzes protein splicing and contains motifs located at the N- and C-terminal regions
that are specific to splicing proteins (5, 6). Consequently, I-CreI is a much smaller protein (163 residues) than
PI-SceI (454 residues). A second reason for the large size
difference between the proteins is that the PI-SceI
endonuclease domain is likely to have evolved by duplication and fusion
of a gene similar to the one that encodes I-CreI. This idea
is supported by the observation of a tandemly repeated sequence and
protein footprinting pattern in homing endonucleases (15).
PI-SceI contains two LAGLIDADG motifs and binds DNA as a
monomer (11), whereas I-CreI has only a single motif and
exists in solution as a homodimer (12).
Structural Comparison of the I-CreI Homodimer and the PI-SceI
Endonuclease Domain--
To elucidate common and distinctive features
of homing endonucleases that contribute to substrate recognition and
cleavage, we first compared the PI-SceI and
I-CreI structures. The PI-SceI structure reveals
that the endonuclease domain (domain II) is folded from two similar
The topology of the secondary structure elements of the two
PI-SceI subdomains is similar to that of the
I-CreI homodimer except for the presence of two additional
helices ( Symmetry Relationships in the I-CreI and PI-SceI Active
Sites--
The overlap between the two structures extends to the
conserved Asp residues at the C-terminal end of the LAGLIDADG repeats that comprise part of the active site. In our alignment, Asp-218 and
Asp-326 of PI-SceI superimpose with Asp-20 and Asp-20' in the two monomers of the I-CreI structure with a root mean
square of less than 1 Å between both pairs of C
A new finding that results from the structural comparison is that
Lys-98 and Lys-98' of the two I-CreI monomers superimpose on
Lys-301 of the PI-SceI N-subdomain and the
pseudosymmetrically related Lys-403 in the C-subdomain (Fig. 1). The
symmetry relationship of Lys-301 and Lys-403 has not been previously
reported. The closest distance between the pair of lysines in
PI-SceI is about 15 Å, which is very similar to the 14-Å
distance that separates the identical pair in the I-CreI
homodimer. The Lys residues are located outside the overlapped
secondary structural elements used to perform the structural comparison
and, thus, deviate in their C Site-directed Mutagenesis of Lys-403--
To elucidate the role of
Lys-403 in the PI-SceI reaction pathway, variant
PI-SceI proteins containing alanine and arginine substitutions were expressed, and purified proteins were assayed for
DNA cleavage and binding activities. As the purification properties of
the K403A and K403R proteins were indistinguishable from those of
wild-type PI-SceI, gross changes in conformation of either mutant protein are unlikely. In kinetic experiments performed in the
presence of Mg2+, the K403A protein is more than 50-fold
less active than wild-type PI-SceI, whereas the K403R
protein is about 5-fold less active (Table
I). Amino acid substitutions at Lys-301
and Lys-403 do not have identical effects because the K301A variant is
inactive, whereas the K403A protein is partially active (Ref. 9 and
Table I). However, for both the Lys-301 and Lys-403 mutant proteins, an
increase in the level of cleavage activity is observed when manganese
is replaced with magnesium in the cleavage buffer, which is known to
increase the enzymatic activity of wild-type PI-SceI (Table
I). It is also evident from our data that the K301R and K403R variants
are more active than the respective K301A and K403A proteins,
suggesting that the positive charge at these positions is critical for
activity.
Gel mobility shift experiments indicate that the large decrease in
cleavage activity of the K403A protein is not because of reductions in
DNA binding. Fig. 2 shows that the upper
and lower protein-DNA complexes that are generated by wild-type
PI-SceI are also apparent for the K403A and K403R proteins,
and the measured level of binding is not markedly different from that
for wild-type PI-SceI.1 The
ratio of upper to lower complex is slightly higher for the Lys-403
mutant proteins relative to wild-type PI-SceI. The large reduction in catalytic activity of the Lys-403 variants coupled with
their near wild-type binding behavior is consistent with a role for
Lys-403 in catalysis. In contrast, no conclusions about the role of
Lys-301 in catalysis can be drawn because of the fact that
nonconservative substitutions at Lys-301 eliminate substrate binding to
region I. Fig. 2 shows that as reported previously, no upper complex is
produced by the K301A protein, and the K301R variant yields an upper
complex that migrates faster than that of wild-type PI-SceI
(9).
The two lysines (Lys-98 and Lys98') in I-CreI that
superimpose on Lys-301 and Lys-403 occur in two identical monomer
subunits and necessarily have identical functions. Consistent with the finding that Lys-301 and Lys-403 are critical for DNA cleavage activity, substitution of Lys-98 and Lys-98' of I-CreI with
glutamine inactivates the protein in a genetic system in
vivo (17). However, no biochemical studies have been performed,
and the basis of the phenotype is unknown (17). Moreover, because
I-CreI is a homodimer, both lysines are necessarily altered
in the mutant, and analysis of proteins with single substitutions at
these positions will require engineering differentially tagged monomers
as has been done for EcoRV (18). It cannot be concluded that
Lys-301 and Lys-403 of PI-SceI have identical functions
given that proteins with mutations at these positions behave
differently. In PI-SceI, where the two monomer subunits are
fused, the two lysines occur in different local environments and may
have evolved somewhat different functions during evolution. If indeed
Lys-301 and Lys-403 have identical functions, the different binding
behaviors of the K301A and K403A proteins could be because of differing
abilities of the protein to accommodate the substituted amino acids at
each position. Lys-403 is located in the middle of the large loop
connecting
A critical role in the homing endonuclease reaction pathway for two
symmetrically related positively charged residues would be reflected by
their conservation among the LAGLIDADG protein family. Indeed,
conserved lysine residues analogous to Lys-301 have been reported in
Block D of inteins (5, 6), and an alignment based on a Hidden Markov
model indicates that conserved lysines and arginines occur at or near
this position in virtually all LAGLIDADG proteins (19). By contrast,
none of the alignments specifically identified Lys-403 of
PI-SceI as a conserved residue. The Hidden Markov model
alignment did identify a conserved lysine in some of the homing
endonucleases only a few residues removed from Lys-403, and we presume
that this residue plays an analogous role in these enzymes (19). To
illustrate the relative positions of the symmetrically related acidic
and basic residues at the putative active site, an alignment of
PI-SceI, the related yeast Ho (F-SceII)
endonuclease, and I-CreI is shown in Fig.
3. Conservation of Lys-403 may not have
been detected in sequence alignments because this residue occurs in a
loop whose sequence may have diverged significantly.
Architecture of the Endonucleolytic Active Site--
Does
PI-SceI use two active sites to effect double strand
cleavage or a single site that acts sequentially? Our finding of two
symmetrically related lysines is consistent with a two-active-site model. In this scenario, the two active sites would be comprised of
Lys-301 and Asp-218 and of Lys-403 and Asp-326, respectively. The two
aspartic acids either act together to bind a single metal ion that is
shared by both active sites or bind two metal ions, one for each active
site, in conjunction with other acidic residues, the polypeptide
backbone carbonyl oxygens, or phosphate oxygens. According to this
model, each I-CreI monomer subunit contains a single active
site, as has been suggested previously (13, 17). If PI-SceI
contains two independent active sites, it might be expected that they
could be uncoupled by mutation to produce a nicking activity. However,
no nicking activity is apparent for the K301A and K403A mutant
proteins.2 Furthermore, no
nicking occurs when either Asp-218 or Asp-326 is substituted with
alanine (8), although this result might be expected if these residues
function to bind metal ion(s) required by both sites. These
observations suggest that either the two putative sites are tightly
coupled and cannot be separated (11) or there is only a single active
site in PI-SceI that cuts both strands.
The architecture of the PI-SceI and I-CreI active
sites can be compared with that of the two active sites in homodimeric
restriction endonucleases, each of which contains at least two acidic
residues that chelate a metal ion and a lysine whose function is
unclear (20). Substitution of the acidic residues in restriction
endonucleases that bind a catalytic metal ion eliminates activity (20),
and similar results are observed when Asp-218 and Asp-326 substitutions are made in PI-SceI (8). Whether the conserved lysine
residues in both families of enzymes are functionally analogous is less certain. Lys-403 of PI-SceI may be involved in catalysis
because mutations at this position reduce cleavage activity but do not affect DNA binding. However, the residue is unlikely to play an essential role in the catalytic reaction mechanism, such as generating the activated water molecule, because a larger reduction in activity would have been expected for the mutant enzymes, but it may still function in other capacities, such as helping to neutralize accumulated negative charge on the presumed pentavalent transition state or binding
to the cleavage products. Similarly, an alanine substitution at the
conserved Lys-92 of EcoRV reduces cleavage activity (21). A
key difference between EcoRV and PI-SceI is that
arginine substitution at Lys-403 of PI-SceI yields a protein
with near wild-type activity, but a K92R mutant of EcoRV is
inactive (22). Thus, additional mechanistic information for both the
restriction enzymes and the homing endonucleases will be required to
determine whether the lysine residues are functionally analogous.
Besides the conserved acidic and basic residues discussed above,
I-CreI residues Gln-47, Arg-51, and Arg-70 and their
symmetric partners have also been implicated in the catalytic pathway
by mutagenesis experiments (17). Similar studies of the related homodimeric endonuclease I-CeuI identified Gln-93 and 93',
which are homologues of Gln-47 and 47' in I-CreI, as
critical amino acids (23). In PI-SceI, Asp-229 occupies a
similar position in an analogous We are grateful to Michael Crist for useful
discussions and excellent technical assistance.
*
This work was supported by National Institutes of Health
Grant GM50815 (to F. S. G.) and National Institutes of Health
Training Grant GM08280 (NIGMS) to the Houston Area Molecular Biophysics Program (to X. D.) and the Howard Hughes Medical Institute (F.A.Q).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 the memory of Dr. Josephine G. Gimble.
§
To whom correspondence should be addressed: Center for
Macromolecular Design, Institute of Biosciences and Technology, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7605; Fax: 713-677-7641; E-mail: fgimble{at}ibt.tamu.edu.
1
M. Crist and F. S. Gimble, unpublished results.
2
D. Hu and F. S. Gimble, unpublished results.
3
M. Crist and F. S. Gimble,
unpublished results.
Identification of Lys-403 in the PI-SceI Homing
Endonuclease as Part of a Symmetric Catalytic Center*
§,, and Center for Macromolecular Design,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-helices that are tightly packed. Two conserved acidic residues (Asp-218 and Asp-326) are located at the C termini of
these two helices and play a critical role in the catalytic mechanism
of the enzyme because substitution of these amino acids with alanine
eliminates cleavage activity but permits substrate binding (8).
Furthermore, mutagenesis experiments indicate that a conserved lysine
(Lys-301), which is 6 Å distant from Asp-218 and occurs in another
conserved motif (Block D), is also critical for catalytic activity (7,
9). We remarked previously that the spatial arrangement of the Asp-218,
Asp-326, and Lys-301 residues resembles that of residues at the active
sites of several restriction enzymes and suggested that the different
endonucleases employ similar reaction mechanisms (7).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
/
substructures (identified as N- and C-subdomains) that are
related by an approximate or pseudo 2-fold symmetry (7).
I-CreI is structurally similar to only the endonuclease domain of PI-SceI as is evident from superimposing the C
atoms of the endonuclease domain onto those of the I-CreI
homodimer (Fig. 1, a-c). The
two PI-SceI subdomains and the I-CreI homodimer form a saddle-shaped structure with the underside lined
by the two sheets and the convex upper surface topped by helices. Models of DNA docked to both proteins indicate involvement of the saddle in DNA binding (7, 13). The length of the saddle in
PI-SceI is about 40 Å, whereas that in I-CreI is
about 70 Å. This size difference is because of a long extended
structure in the I-CreI homodimer comprised of 1 and 2
strands and a connecting loop that form parts of the two diagonally
opposite sides of the homodimer saddle that is significantly shorter in
the analogous structures in the N-subdomain (a short loop and 14)
and C-subdomain (19 and 20) of PI-SceI (Fig.
1a-d). The length of the groove formed by the underside of
the I-CreI saddle is sufficient to bind a roughly 20-base
pair pseudo-symmetric DNA homing sequence (13). In contrast, the groove
length in PI-SceI is sufficient to cover only about 12 base
pairs of DNA, far shorter than the required 31 base pairs. Fewer
binding contacts may be required by the PI-SceI endonuclease
domain because additional contacts are made by the protein-splicing
domain (9, 16). We speculate that PI-SceI lost the extended
saddle architecture present in I-CreI once it became
associated with a protein splicing domain that acquired DNA binding
activity.
View larger version (50K):
[in a new window]
View larger version (34K):
[in a new window]
Fig. 1.
Superpositioned structures of the
endonuclease domain of PI-SceI (blue) and the
homodimer structure of I-CreI (yellow and
gold). a, a stereo view of the overlapped
structures viewed down the 2-fold symmetry. The saddles in both
proteins are upside down. The first or N-subdomain (top) and
the second or C-subdomain (bottom) of the PI-SceI
nuclease domain are overlapped with one subunit (yellow,
top) and the other subunit (gold,
bottom), respectively, of the I-CreI homodimer
structure. The residues colored cyan in PI-SceI
are the two pairs of residues related by a pseudo 2-fold symmetry:
Asp-218 and Lys-301 in the N-subdomain and Asp-326 and Lys-403 in the
C-subdomain. The two symmetry-related residues, Asp-20 and Lys-98, in
both monomers of I-CreI are colored red.
b, same as in a but rotated by 90 Å about a
horizontal axis for a view down the groove. The N-subdomain of
PI-SceI is in the front. c, identical to
b but rotated by 180 Å about a vertical axis to bring the
C-subdomain of PI-SceI in the front. d, schematic
diagram of the secondary structures of PI-SceI
(left) and I-CreI homodimer (right).
The lengths of the -helices (green cylinders) and strands (flat pink arrows) are approximately to scale.
Disordered loops are indicated by broken lines. In
PI-SceI, the loop that follows 4 makes only one hydrogen
bond with the adjacent 14, insufficient to qualify as a strand,
although it overlaps with 1 of I-CreI.
7 and 8) at the C-terminal end of I-CreI
(Fig. 1d). Of the 200 residues that are topologically
similar between the PI-SceI endonuclease domain and the
I-CreI homodimer, 102 C atoms overlap with a root mean
square of 2 Å and a sequence similarity of 27%. Despite the topological similarity, the extent of overlap of the I-CreI
homodimer with the two subdomains differs considerably; of the 102 overlapped residues, 67 are made with the C-subdomain and 35 with the
N-subdomain. Interestingly, the number of superimposed residues made
with the C-subdomain is similar to that in the overlap between the two PI-SceI subdomains (63 residues with a root mean square of
1.7 Å). This supports the idea that the endonuclease domain of
PI-SceI evolved by tandem duplication of a common ancestor
of the I-CreI monomer and of one PI-SceI
subdomain. Once the gene fusion occurred in PI-SceI, each
subdomain could evolve independently to optimize substrate specificity.
The structural comparison suggests that the N-subdomain diverged
further from the evolutionary ancestor than the C-subdomain because it
is far less similar to the I-CreI subunit. The ability of
the two subdomains to evolve at different rates, which is not possible
in a homodimeric protein, may have permitted PI-SceI to
recognize an asymmetric substrate.
atoms (Fig.
1a-c). Substitution of any of these acidic residues with
asparagine eliminates cleavage activity (8, 17). Taken together, these
observations strongly suggest that these Asp residues play the same
functional role in the endonuclease activity of both proteins, which
may be to chelate the essential Mg2+ ion co-factor(s).
However, the coordination of the cation is unknown in both structures.
position (2.4 Å between Lys-301 and
Lys-98 and 3.5 Å between Lys-403 and Lys-98'). However, all their side
chains point from the same direction to the well overlapped active-site
Asp residues (Fig. 1).
Cleavage activity of wild-type and mutant PI-SceI proteins
View larger version (91K):
[in a new window]
Fig. 2.
DNA binding properties of wild-type and
mutant PI-SceI proteins. Autoradiogram of a native 7%
acrylamide gel showing the protein-DNA complexes (UC, upper
complex; LC, lower complex; UB, unbound) obtained
using wild-type PI-SceI or the K301A, K301R, K403A, and
K403R mutant proteins (0.7 nM) in a gel mobility shift
experiment.
9 of domain II with 23 of domain I, whereas Lys-301 is
located at the end of 18, which is part of the two-stranded short
sheet connecting the two subdomains of domain II. Because strand is not as flexible as the loop, the mutation of Lys-301 to
arginine may be unable to be positioned correctly to achieve full
catalytic function like the K403R mutant.
View larger version (13K):
[in a new window]
Fig. 3.
Alignment of amino acid sequences from
selected regions of PI-SceI, Ho, and I-CreI
endonucleases. The two acidic residues, Asp-218 and Asp-326, and
two basic residues, Lys-301 and Lys-403, that are presumed to comprise
the PI-SceI active site(s) and the analogous residues in Ho
and I-CreI are indicated by reverse lettering.
Sequences from I-CreI have been manually aligned with blocks
C, D, and E identified in the PI-SceI and Ho proteins (6).
Identification of Lys-98 in I-CreI and Lys417 in Ho as
analogues of Lys-403 in PI-SceI was accomplished by
structural and sequence alignments, respectively. The position in the
protein of the last amino acid in each block is indicated to the right
of the block.
structure as Gln-47, but the side
chains of the two residues do not overlap because the carboxyl group of Asp-229, unlike the Gln-47 amide, points away from the active site (9).
We suggested previously that Asp-229 may be involved in catalysis
because a D229A mutant protein is reduced >500-fold in cleavage
activity but is only slightly reduced in substrate binding (9).
Moreover, there is evidence that an acidic residue is conserved at or
near this position (19). It is possible that Asp-229 functions to
coordinate a metal ion in conjunction with Asp-218 as part of one
active site, but if this is true, rotation of the Asp-229 side chain
and additional conformational changes would be required when the
protein binds to the substrate. PI-SceI residue Thr-341 is
related to Asp-229 by pseudosymmetry and overlaps Gln-47' in
I-CreI. A T341A mutant protein does not form an upper complex in gel mobility shift experiments, and this reduced binding may
account for the ~11-fold reduction in catalytic activity of the
protein relative to wild-type
PI-SceI.3 However,
it is unlikely that Thr-341 is involved in binding the metal co-factor
in a second active site because a larger decrease in activity would be
expected for the mutant protein. The side chains of PI-SceI
residues Arg-231 and His-343 are close to, but do not overlap, those of
Arg-51 and -51' in I-CreI. R231A and H343A mutant proteins
are 14- and 5-fold reduced in activity, respectively, and the H343A
protein displays a modest reduction in binding (9). Whether these
residues are functional analogues of I-CreI residues Arg-51
and -51' is unclear. Finally, Arg-70 in one I-CreI monomer
is located in a position similar to Asp-254 or Arg-255 of
PI-SceI, and Arg 70' in the other monomer closely overlaps
with Glu-366 in PI-SceI. However, these PI-SceI
residues are unlikely to be Arg-70 analogues because alanine
substitutions at these positions do not adversely affect
PI-SceI activity. Taken together, these data indicate that
in some cases there are PI-SceI and I-CreI
residues that occur at analogous positions that may play analogous
functions in the cleavage reaction. A structure of a homing
endonuclease bound to its substrate will help support this conclusion.
ACKNOWLEDGEMENT
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Koufopanou and A. Burt Degeneration and Domestication of a Selfish Gene in Yeast: Molecular Evolution Versus Site-Directed Mutagenesis Mol. Biol. Evol., July 1, 2005; 22(7): 1535 - 1538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Posey, V. Koufopanou, A. Burt, and F. S. Gimble Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species Nucleic Acids Res., July 27, 2004; 32(13): 3947 - 3956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bakhrat, M. S. Jurica, B. L. Stoddard, and D. Raveh Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast Genetics, February 1, 2004; 166(2): 721 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Thion, E. Laurine, M. Erard, O. Burlet-Schiltz, B. Monsarrat, J.-M. Masson, and I. Saves The Two-step Cleavage Activity of PI-TfuI Intein Endonuclease Demonstrated by Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry J. Biol. Chem., November 15, 2002; 277(47): 45442 - 45450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Chevalier and B. L. Stoddard Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility Nucleic Acids Res., September 15, 2001; 29(18): 3757 - 3774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Hu, M. Crist, X. Duan, F. A. Quiocho, and F. S. Gimble Probing the Structure of the PI-SceI-DNA Complex by Affinity Cleavage and Affinity Photocross-linking J. Biol. Chem., January 28, 2000; 275(4): 2705 - 2712. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |