(Received for publication, October 13, 1994; and in revised form, January 6, 1995)
From the
The PI-SceI endonuclease from yeast belongs to a
protein family whose members contain two conserved dodecapeptide motifs
within their primary sequences. The function of two acidic residues
within these motifs, Asp and Asp
, was
examined by substituting alanine, asparagine, and glutamic acid
residues at these positions. All of the purified mutant proteins bind
to the PI-SceI recognition site with the same affinity and
specificity as the wild-type enzyme. By contrast, substituting alanine
or asparagine amino acids at the two positions completely eliminates
strand cleavage of substrate DNA, whereas substitution with glutamic
acid markedly reduces the cleavage activity. Experiments using nicked
substrates demonstrate that the wild-type enzyme shows no strand
preference during cleavage. These results are consistent with a model
in which both acidic residues are part of a single catalytic center
that cleaves both DNA strands. Furthermore, substrate binding by
wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion
attack at the cleavage site while binding by the alanine-substituted
proteins either stimulates this attack significantly less or protects
the DNA at this position. These finding are discussed in terms of
possible reaction mechanisms for PI-SceI-mediated
endonucleolytic cleavage.
The PI-SceI endonuclease (formerly named VDE) ()cleaves the yeast genome at a single location and
initiates a gene conversion process that results in the transfer of the
endonuclease gene to other yeast strains(1, 2) . The
endonuclease is initially translated as part of a precursor protein and
occurs as an internal protein sequence or ``intein'' that is
flanked by two external protein sequences or ``exteins'' (for
nomenclature information see(3) ). Through a process termed
protein splicing, the PI-SceI intein excises itself from the
precursor and joins together the two exteins to generate a second
unrelated protein(4, 5, 6) . The excised
PI-SceI protein belongs to a family of related DNA
endonucleases that share common biochemical and structural properties
(for reviews, see (7) and (8) ). These enzymes cleave
DNA within their recognition sequences to leave 4-bp 3`-OH overhangs,
require Mg
ion as a co-factor, and, unlike the
prokaryotic restriction endonucleases, recognize sites that are longer
than 18 bp in length that contain no obvious dyad symmetry. Another
distinguishing feature of these endonucleases are two conserved
12-residue peptide sequences that are spaced approximately 100 amino
acids apart ((9-11), see Fig. 1). It is unlikely that
these dodecapeptide motifs are responsible for mediating substrate
specificity since each endonuclease recognizes and cleaves at a
different site. Instead, the residues within these motifs may comprise
part of the catalytic center.
Figure 1:
Protein sequence alignment
of the dodecapeptide motifs within related endonucleases (modified and
updated from(8) ). Conserved amino acids are indicated by white on black lettering, and the number of intervening
residues between the motifs is indicated. Amino acids are represented
by the single-letter code. In the consensus sequences, specific amino
acids are indicated if they are invariant or if they represent 50%
of the total; hydrophobic residues are indicated by O (when
65% of
the total, regardless of specific amino acid content); acidic residues
are indicated by B (
90% of total), and X represents any residue. The I prefix indicates proteins involved
in intron homing(40) ; the PI prefix denotes proteins generated
from protein splicing events(3) . Proteins marked with an asterisk are generated by protein splicing, but have not been
shown to be endonucleases. The arrows indicate the aspartic
acid residues in PI-SceI that have been substituted by
site-directed mutagenesis. Data base accession numbers for the
sequences: Ctr VMA, M64984; PI-PspI, U00707, D29671; Mle recA, X73822; the remainder are given in (8) .
To examine whether the two dodecapeptide motifs are involved in the cleavage reaction and/or DNA binding, we used site-directed mutagenesis to substitute glutamic acid, asparagine, or alanine at the positions of two conserved aspartic acid residues within the motifs. Here, we show that all of the purified mutant proteins bind to DNA normally, but only the glutamic acid substituted enzymes cleave DNA. We conclude from these experiments that acidic amino acids are likely to be required at each of these two positions for cleavage to occur and that the two aspartic acid residues comprise a single catalytic center that cleaves both DNA strands. Previously, it has been reported that substitution of an analogous aspartic acid residue within the PI-TliI endonuclease, a member of the same protein family as PI-SceI, eliminates catalytic activity(12) . In this paper, we have significantly extended this analysis by examining the roles of both dodecapeptide motifs and by showing for the first time that the strand-scission and DNA binding activities can be effectively uncoupled for this class of enzymes.
Figure 5:
Cleavage activity assays of wild-type and
mutant (D218A, D326A) PI-SceI proteins using
full-length and nicked substrates. A, autoradiogram of a
denaturing gel showing the cleavage products obtained following
digestion of various synthetic duplex oligonucleotides with wild-type
or mutant PI-SceI proteins. Cleavage reactions were assembled
as described under ``Experimental Procedures'' and were
incubated at 37 °C for 1 h. An equal volume of loading buffer was
added to each reaction mixture, and the products were resolved by
electrophoresis on a 15% denaturing acrylamide slab gel which was dried
and exposed to x-ray film. The letters above each lane denote
the oligonucleotides (shown in B) that were annealed to form
the substrate for that reaction. An asterisk indicates which
oligonucleotide was labeled at its 5`-end with P in each
reaction. B, synthetic oligonucleotides (A-F) used to
construct the full-length substrate and fragments used in this
experiment. Cleavage sites on each strand are indicated by arrows.
We overproduced and purified wild-type PI-SceI and
the D218A, D218N, D218E, D326A, D326N, D326E mutants in order to assay
their enzymatic and DNA binding properties in vitro. Each of
the mutant proteins exhibited the same purification behavior as the
wild-type protein. Gel shift experiments were performed to examine
substrate binding by using a 67-bp duplex oligonucleotide that contains
the PI-SceI cleavage/recognition
site(1, 13) . In the absence of Mg ion, a required co-factor for cleavage, the wild-type protein and
each of the mutant proteins binds to the labeled 67-bp probe and yields
a single protein-DNA complex (Fig. 2A). Gel shift
assays were used to measure apparent equilibrium dissociation constants
for each of the proteins (Table 1). These values (average K
2.4 nM) are essentially the same
for the mutant and wild-type endonucleases.
Figure 2:
Gel shift analysis of wild-type and mutant
PI-SceI proteins. A, sequence of the synthetic 67-bp
duplex used as substrate. Oligonucleotides containing the wild-type
recognition sequence were synthesized as described under
``Experimental Procedures.'' CS indicates the
cleavage sites on both strands. The arrows mark the boundaries
of a DNase I footprint (Fig. 3). B, gel shift analysis
in the absence of Mg ion. Approximately 5-10
fmol of 5`-end-labeled 67-bp duplex substrate were incubated with
wild-type or mutant PI-SceI protein (13 nM) in gel
binding buffer (25 mM Tris-HCl (pH 8.5), 100 mM KCl,
10% glycerol, 50 µg/ml bovine serum albumin, and 2.5 mM 2-mercaptoethanol) that did not contain Mg
ion.
The unbound and bound DNA species were separated by electrophoresis on
a 7% nondenaturing gel and migrated to the positions labeled Free and Complex, respectively. The proteins used are
indicated above each lane. The designations top and bottom refer to which strand shown in A is end-labeled in the
DNA duplex. C, gel shift analysis was performed as in B except that the gel binding buffer contained 2.5 mM MgCl
. The two cleavage products migrate to the
positions labeled F1 and F2. These gels have been
overexposed in order to show the low levels of cleavage product. The
faint bands seen in the control reactions that co-migrate with the
complex are due to spillover between lanes.
Figure 3:
DNase I protection analysis of wild-type
and mutant PI-SceI proteins. A, footprint of the
PI-SceI cleavage/recognition site. Purified wild-type or
mutant PI-SceI protein (5 nM total concentration)
were incubated with end-labeled 67-bp duplex substrate (25,000 cpm,
10-20 fmol) as described under ``Experimental
Procedures.'' Digestion with DNase I (5 µl of a 3 µg/ml
stock) was performed for 5 min at 25 °C. The reaction products were
resolved on 14% denaturing gels (7 M urea, 1 TBE,
acrylamide:bisacrylamide, 19:1) which were subsequently dried and
exposed to x-ray film. Lanes are designated as in Fig. 2; the
lane marked A + G contains products from a Maxam-Gilbert
A + G reaction(19) . CS indicates the cleavage
site; brackets represent protected areas. B,
schematic of DNase I footprint (indicated by brackets)
relative to the cleavage site. These data are representative of the
results from two different experiments, which were
indistinguishable.
DNase I protection experiments were used to establish whether the mutant proteins bound to the recognition sequence with the same binding pattern as wild-type PI-SceI. Fig. 3shows that the wild-type protein uniformly protects a 35-bp region on each strand from DNase I cleavage. This region overlaps the cleavage site and is nearly coincident with a 30-bp region that was determined previously to be the minimum region required for PI-SceI-mediated cleavage(13) . Under the conditions used in this assay, cleavage products are not observed because of the slow rate of cleavage by the enzyme. Each of the mutant proteins yields a protection pattern that is identical to that of the wild-type protein (Fig. 3A). The amount of protection produced by the D218E and D326A proteins at the concentration used is approximately 2-fold to 3-fold less than that of wild-type PI-SceI even though the equilibrium dissociation constants determined by gel-shift analysis are very similar. This difference may be due to the fact that the DNase I experiments are performed in solution, whereas the gel-shift experiments require passage of the protein-DNA complex through the gel matrix.
Similar results are observed when the wild-type and mutant
enzymes are used to cleave a linearized plasmid substrate that contains
a single copy of the PI-SceI cleavage/recognition site (Fig. 4). The wild-type enzyme cleaves the substrate nearly
completely, the D326E mutant protein generates at least 6-fold less
cleavage product and the other mutants yield no products. Previously,
we have shown that replacing the Mg ion in the
reaction buffer with Mn
ion causes PI-SceI
to cut DNA at non-cognate sites and to cleave the cognate site at
faster rates (1) . When Mn
ion is used in
these experiments, the D218E mutant enzyme functions to cleave the
substrate (Fig. 4). Furthermore, the amount of cleavage by the
D326E protein in the presence of Mn
ion is greater
than in the presence of Mg
ion. Although we have not
mapped the cleavage sites created by the glutamic acid mutants at the
nucleotide level, it is likely that they are the same as or similar to
those created by the wild-type enzyme as judged by the size of the
cleavage products.
Figure 4:
Cleavage activity assays of wild-type and
mutant PI-SceI proteins using a linearized plasmid substrate.
Cleavage of plasmid pBSVDEX (13) linearized with XmnI
was performed as described under ``Experimental Procedures.''
Approximately 10 units of wild-type PI-SceI were used where
one unit is sufficient to cleave 100 ng of linearized substrate in 1 h
at 37 °C. Aliquots of each reaction were separated by
electrophoresis in 1 TBE buffer on a 0.9% agarose gel, which
was stained with ethidium bromide and photographed.
PI-SceI-mediated cleavage generates 2.6- and 1.1-kilobase
products. The reactions included either 2.5 mM MgCl
or 2.5 mM MnCl
as indicated above the
gel.
The alanine- and asparagine-substituted mutant proteins lack double-strand cleavage activity, but it is possible that they are able to cleave single strands of the substrate (i.e. ``nick'' the DNA). We tested for this activity by incubating the wild-type enzyme and the two alanine mutants with end-labeled substrates and by analyzing the cleavage products on denaturing gels. The wild-type protein cleaves the full-length substrate and generates the two expected cleavage products (Fig. 5). Unlike the experiment shown in Fig. 2C, equal amounts of both cleavage products are observed here because the denaturing gel system being used prevents one of the products from remaining bound to the protein. In contrast to these results, the two alanine mutants do not produce either cleavage product, indicating that both proteins are incapable of nicking the DNA on either strand. By using nicked substrates (see ``Experimental Procedures''), we also examined whether these mutants cleave the second strand if the first strand is already cut. The wild-type enzyme effectively cleaves the second strand of both nicked substrates, whereas neither alanine mutant cleaves either nicked DNA substrate (Fig. 5). In sum, these results demonstrate that the alanine mutant proteins are unable to cleave either strand of the substrate.
Figure 6:
Hydroxyl radical protection analysis. A, footprint of the PI-SceI cleavage recognition
site. Hydroxyl radical reactions were performed as described under
``Experimental Procedures,'' and the modified DNA products
were separated by electrophoresis on a 12% denaturing gel (7 M urea, 1 TBE). Lane AG, products from a
Maxam-Gilbert DNA sequencing reaction; lane F, free duplex
treated with [Fe(II)EDTA]
Surprisingly, the protection patterns near the cleavage site are
very different for the wild-type and mutant proteins. In the presence
of wild-type PI-SceI, some positions near the cleavage site
are hypersensitive to hydroxyl radical cleavage. On the top strand, the
cytosine adjacent to the cleavage site (C) is
hypersensitive to cleavage, and nucleotides G
,
G
and G
are also hypersensitive,
but to a lesser degree. On the bottom strand, the two cytosines that
border the cleavage strand (C
and
C
) are equally hypersensitive. No hypersensitivity
is observed if either hydrogen peroxide or
[Fe(II)EDTA]
We have constructed six variants of the PI-SceI endonuclease whose behaviors suggest that the enzyme uses a single catalytic center to effect strand cleavage. Substitutions were made for two aspartic acid residues that occur in two dodecapeptide motifs that define a family of site-specific DNA endonucleases and RNA maturases (7, 8) . We found that substitution of either aspartic acid residue with asparagine or alanine effectively uncouples the DNA binding and strand scission activities and results in mutant proteins that bind to the substrate DNA with the same affinity as the wild-type protein, but fail to cleave it. Substitution with glutamic acid results in proteins with decreased cleavage activity, suggesting that the acidic nature of the side chain plays a critical role in the cleavage mechanism. Taken together, our results provide strong evidence that the dodecapeptide motifs contain the active site residues for the cleavage reaction.
Two models for PI-SceI-mediated cleavage are possible, either the enzyme contains a single active site that cuts both strands or two active sites, each of which cuts one DNA strand. If there are two sites and each is comprised of the amino acids within a single dodecapeptide motif, it might be expected that disabling one of them would result in an enzyme that is still able to nick the DNA on one strand. However, we show here that introducing mutations at either of the two conserved aspartic acid residues prevents the enzyme from cleaving the duplex DNA on either strand. It could still be argued, however, that each single mutation disrupts two active sites by globally perturbing the protein conformation. This is unlikely because 1) the behaviors of the mutant proteins during purification are indistinguishable from that of wild-type PI-SceI enzyme, and 2) the binding affinities of the mutant and wild-type enzymes for the substrate DNA are the same. Alternatively, nicking activity may not be observed because the double-strand cleavage reaction involves two sequential single-strand cleavage steps that occur in a fixed order. If the first step is prevented by mutation, the second step cannot occur. This possibility was tested in the case of the FokI restriction endonuclease by using a nicked substrate that was identical in structure to a duplex substrate where the first strand had already been cleaved(23) . When we performed similar experiments, the PI-SceI mutant enzymes, like FokI(23) , failed to cleave the nicked substrates. These data are consistent with a single active site model.
How might PI-SceI effect
cleavage of its substrate using a single active site? Examining the
similarities between PI-SceI and the FokI enzyme,
which is a type IIS restriction endonuclease(31) , may provide
an answer. Both enzymes recognize asymmetric sequences, are monomers in
solution and require Mg co-factor for cleavage, but
not for binding(13, 32) . More importantly, each of
two aspartic acid residues within FokI are required for DNA
cleavage activity, but not for DNA binding(23) . These two
aspartic acids may be part of a single active site within FokI
which cleaves both strands(23) . Following cleavage of the
first strand, a conformational change in the enzyme or the DNA
substrate could be required to allow cleavage of the second strand to
proceed(23) . A mechanism of sequential strand scission could
also by used by the PI-SceI enzyme. However, even if the
reaction mechanisms are similar for FokI and PI-SceI,
their strategies for recognizing and binding to DNA are likely to be
different. The FokI endonuclease can be divided into a DNA
binding domain and a catalytic domain(33) , but there is no
evidence that the same is true for PI-SceI. In addition, the
recognition sequence for FokI is 5 bp long, whereas the
minimum sequence required for PI-SceI-mediated cleavage is in
excess of 30 bp.
The exact role of the two aspartic acid residues in
PI-SceI during catalytic cleavage is unclear, but it may be
similar to the function of the conserved acidic residues that are found
in the EcoRI, EcoRV, FokI, BamHI,
and PvuII restriction
endonucleases(22, 23, 25, 26) .
Substantial structural and mechanistic information exists in the case
of the EcoRI and EcoRV proteins that suggests a
function for these acidic residues. In these proteins, the two acidic
residues as well as a conserved lysine are positioned almost
identically with respect to the scissile phosphodiester
bond(34) . In one model, the acidic residues are thought to
bind to the Mg ion at the active site which is
believed to help stabilize the negative charges on the pentavalent
transition state following nucleophilic attack by an activated water
molecule. Although the sequences of the dodecapeptide motifs within
PI-SceI are dissimilar from the motifs that occur in these
restriction endonucleases, the acidic residues may play similar roles
in chelating a Mg
ion. However, even if the two
aspartic acid residues have a similar function in PI-SceI,
they are not equivalent since the Asp
and Asp
mutant proteins behave differently in the cleavage assay and
during hydroxyl radical footprinting. Little is known about the
function of the conserved hydrophobic and glycine residues within the
dodecapeptide motifs. The singular ability of glycine amino acids to
adopt conformations not available to other residues may be critical for
orienting the aspartic acid side chains during catalysis.
The
hypersensitivity to hydroxyl radical attack that occurs upon binding of
wild-type PI-SceI to its substrate was unexpected. This
hypersensitivity is markedly reduced when the alanine mutants bind to
the substrate; in fact, the D326A protein significantly protects this
region. The hypersensitivity is clearly the result of hydroxyl radical
or hydroxide ion attack and not due to the normal
PI-SceI-mediated cleavage reaction, since this effect is only
observed if all of the components of the Fenton reaction are present.
Two models can be used to explain the source of the observed
hypersensitivity. First, if the aspartic acid residues normally chelate
Mg ion, they may also be able to bind Fe
ion, whose ionic radius is only slightly larger (0.66 Å for
Mg
versus 0.74 Å for
Fe
(35) ), or the
[Fe(II)EDTA]