Figure 4. Sequence alignment of all known group I intron-encoded proteins that are related to I-CeuI. The compared proteins are encoded by introns inserted at sites 1923, 1931, 1951 and 2593 in the LSU rRNA gene and also by mitochondrial introns found in other genes. Identical residues are shown on a black background, while conserved sets of amino acids sharing eight of the 10 features in the property matrix intra.pt of the AMAS program (29) are shown on a grey background. Note that in the calculation of conservation values up to 8% of atypical residues at any position of each group of sequences were ignored. The coordinates refer to the I-CeuI protein. The letters above the alignment indicate the single amino acid substitutions that are described in Table 1. The proteins known to display endonuclease activity are designated according to Dujon et al. (32); the introns encoding the other proteins are abbreviated according to Michel and Westhof (35), except that OXI was replaced by COI and that a three-letter code, instead of a two-letter code, was used to designate Chlamydomonas mexicana. All protein sequences are partial; in each sequence, the segment preceding the single or the second copy of the dodecapeptide was deleted. All of the LSU rDNA intron-encoded proteins as well as the PaND4[middot]1-encoded protein contain a single copy of the dodecapeptide, whereas the remaining ones contain two copies of this sequence. Database accession numbers for the sequences: I-CpaI, L36830; CmeLSU[middot]1, L49148; AcLSU[middot]1, s53825; I-CreI, a23091; AcLSU[middot]3, s53827; NcATP6[middot]2, X01075; PaCOI[middot]5, f48327; SpCOI[middot]2, P22190; NiND1[middot]1, s06367; PaND1[middot]4, s06059; PaND3[middot]1, s05654; PaND4[middot]1, s05653; NcND4L[middot]1, s10840; PaND4L[middot]1, s09134; PaND4L[middot]2, S09141; NcND5[middot]1, s10841; PaND5[middot]2, s09143; SsSSU[middot]1, U07553; the remainder are given in the legend of Figure 1.

Analysis of mutations among I-CeuI gene sequences expressed in E.coli

We have previously encountered major problems in recovering stable clones of E.coli producing large amounts of the I-CeuI endonuclease from expression plasmids. This endonuclease proved highly toxic to E.coli when we expressed the I-CeuI gene using the trc promoter expression vectors pKK233-2 and pTRC-99A (13 ) as well as other expression plasmids (15 ): high frequency of frameshift mutants were observed, extremely low levels of expression were obtained, and growth of E.coli was found to be greatly affected. As the I-CeuI endonuclease has been shown to cleave the E.coli chromosomal DNA specifically within the rRNA operons (16 ), the toxicity of I-CeuI is very likely to be a consequence of such cleavage. Assuming that this hypothesis is valid, it should be possible to easily isolate mutants in the I-CeuI gene by searching for clones whose growth is not impaired by the expression of this gene.

With the goal of isolating such mutants, we undertook the construction of a library of E.coli clones expressing the I-CeuI gene. This gene was amplified by PCR using conditions that are expected to yield a frequency of mutations of 0.25-0.4% (36 ) and it was cloned into the NcoI and BamHI sites of the T7 expression plasmid pET-30a(+) in order to fuse the I-CeuI gene in-frame with the sequences coding for the His- and S-tags. The recombinant plasmids were then introduced into the E.coli strain NovaBlue (DE3) containing a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter and transformants were selected in the absence of IPTG. Even under these conditions in which transcription from both the lacUV5 and T7 promoters is inhibited, the I-CeuI endonuclease was produced in amounts that are lethal for E.coli cells. The efficiency of transformation was several orders of magnitude lower than expected, and all 133 recombinant clones analyzed were found to display mutations in their I-CeuI gene sequence. Because we also failed to recover the wild-type form of the I-CeuI gene by transforming a NovaBlue E.coli strain lacking the T7 RNA polymerase gene with our plasmid construct, it is likely that transcription of the I-CeuI gene occurred from cryptic promoters present on the pET30(a+) plasmid.

Table 1 . Nature and sites of the mutations responsible for the single amino acid changes in I-CeuI variants

Codon	Codon position	Base change	Type of mutationa	Amino acid change	Independent clones
58	1	TTT -> CTT	Ts	F -> L	1
59	2	TTA -> TCA	Ts	L -> S	1
61	1	GGT -> AGT	Ts	G -> S	1
61	2	GGT -> GAT	Ts	G -> D	1
62	2	TTT -> TCT	Ts	F -> S	4
64	1	GAA -> AAA	Ts	E -> K	3
64	2	GAA -> GGA	Ts	E -> G	1
64	2	GAA -> GCA	Tv	E -> G	1
64	2	GAA -> GTA	Tv	E -> V	1
66	1	GAA -> AAA	Ts	E -> K	1
66	2	GAA -> GTA	Tv	E -> V	2
66	2	GAA -> GGA	Ts	E -> G	1
67	1	GCT -> ACT	Ts	A -> T	1
68	2	TCT -> TTT	Ts	S -> F	1
70	1	AAT -> GAT	Ts	N -> D	1
72	2	AGC -> AAC	Ts	S -> N	1
74	2	AAA -> AGA	Ts	K -> R	1
80	1	AAA -> GAA	Ts	K -> E	1
86	1	GAT -> AAT	Ts	D -> N	1
88	1	GAA -> AAA	Ts	E -> K	1
89	1	TTC -> CTC	Ts	F -> L	1
90	1	AAT -> TAT	Tv	N -> Y	1
91	2	GTG -> GGG	Tv	V -> G	1
92	1	ACT -> GCT	Ts	T -> A	1
93	2	CAA -> CGA	Ts	Q -> R	3
94	1	CAT -> TAT	Ts	H -> Y	1
94	2	CAT -> CGT	Ts	H -> R	2
96	1	AAT -> GAT	Ts	N -> D	1
97	1	GGG -> AGG	Ts	G -> R	1
108	2	TTT -> TCT	Ts	F -> S	1
111	1	GGG -> AGG	Ts	G -> R	1
111	2	GGG -> GAG	Ts	G -> E	1
112	1	CGT -> TGT	Ts	R -> C	1
113	2	ATT -> AAT	Tv	I -> N	1
116	1	AAA -> GAA	Ts	K -> E	1
116	1	AAA -> CAA	Tv	K -> Q	1
121	2	GCA -> GTA	Ts	A -> V	1
122	1	ACT -> GCT	Ts	T -> A	1
141	1	TAT -> AAT	Tv	Y -> N	1
152	1	GAA -> CAA	Tv	E -> Q	1
152	1	GAA -> AAA	Ts	E -> K	1
153	1	AAA -> GAA	Ts	K -> E	1
186	1	TGG -> CGG	Ts	W -> R	4
186	2	TGG -> TTG	Tv	W -> L	1
189	2	ATG -> AAG	Tv	M -> K	1
190	1	CGT -> TGT	Ts	R -> S	1
192	2	CAA -> CGA	Ts	Q -> R	3
200	2	TTT -> TCT	Ts	F -> S	1
200	3	TTT -> TTA	Tv	F -> L	1

^aTs, transition; Tv, transversion.

Sixty eight percent of the recombinant clones (90/133) revealed single- or double-point mutations in the I-CeuI gene, whereas the others displayed frameshift mutations. A total of 66 clones (or 50%) harboured single-point mutations: 58 of these mutations are responsible for amino acid changes at 35 positions, while the remaining ones account for the creation or deletion of stop codons at distinct sites. Considering the five double mutants exhibiting one synonymous and one non-synonymous mutation, a total of 49 variant enzymes showing distinct, single amino acid changes at 37 positions were recovered (Table 1 and Fig. 4 ). All but four of these positions (positions 59, 80, 88 and 152) coincide with sites that are evolutionarily conserved within the I-CeuI protein subfamily (see Figs 3 and 4 ). Most of the amino acid changes (37/49) are found in the portion of the protein occupied by the first 123 amino acids and they are clustered in the regions containing the dodecapeptide, TQH motif and G¹¹¹. At the gene level, the great majority of the substitutions occurred at first and second codon positions, and transitions were found to be more frequent (3.1 times more numerous) than transversions (Table 1 ). Assuming that all mutations were caused by transitions, two amino acid substitutions would be typically predicted at each altered position. Saturation in the number of transition events was observed at only five of the 37 positions that revealed amino acid changes. To reduce the bias in the ratio of transitions and transversions, we tried to amplify the I-CeuI gene in the presence of 0.5 mM MnCl₂; however, these conditions which are known to yield an equal ratio of transitions and transversions and reduce the fidelity of DNA synthesis (36 ) did not allow us to diversify our collection of mutants with single substitutions since the frequency of mutants with multiple mutations rose sharply.

Kinetic properties of I-CeuI variant endonucleases

Each of the I-CeuI variant enzymes is predicted to be altered in its kinetic properties relative to the wild-type form. To verify this prediction and hence confirm that our E.coli expression library can provide valuable information on the structure/function relationships of I-CeuI, we have determined the k_cat and K_m values of nine randomly selected variants showing single amino acid changes at distinct positions (Table 2 ). These enzymes as well as the wild-type form were synthesized in vitro in rabbit reticulocytes lysates and assayed at 37^oC in the presence of 2.5 mM MgCl₂ and various concentrations of a ³³P-labeled DNA substrate. This substrate is the 258 bp PCR product that was used to determine whether the site-1923 intron-encoded proteins are endowed with endonuclease activity. The reaction products were identified and quantified using a phosphorimager. The K_m and k_cat of the wild-type enzyme were found to be 0.9 +- 0.3 nM and 3.7 * 10^-5 s^-1, respectively. This extremely low k_cat value indicates that I-CeuI resembles the endonucleases I-SceI and PI-SceI (an intein-derived enzyme) in turning over very slowly (6 ,37 ). Table 2 shows that all nine variants, except T122A, display k_cat/K_m values that are at least 10 times lower than that of the wild-type. Although the T122A mutant does not differ from the wild-type in the kinetic parameters measured, its cleavage pattern is distinct. It shows a greater preference for the top strand, with the relative amount of the reaction product corresponding to this strand being three times more abundant than that observed for the wild-type enzyme (data not shown). Three variants (E66K, Q93R and K116Q) failed to reveal any detectable cleavage activity even in the presence of a large excess of substrate. Variants H94R and W186R feature the lowest k_cat value, while variant F62S, with a K_m of 25 nM, deviates the most from the wild-type regarding this parameter.

Table 2 . Relative kinetic parameters of nine I-CeuI variant enzymes synthesized in vitro

I-CeuI enzyme	kcat	Km	kcat/Km
Wild-typea	1	1	1
F62S	0.5	28	0.02
E64V	0.5	7	0.07
E66K	<0.01	ND	<0.01
Q93R	<0.01	ND	<0.01
H94R	0.1	2	0.05
K116Q	<0.01	ND	<0.01
T122A	1	1	1
W186R	0.2	2	0.10
F200L	0.4	6	0.07

^aThe I-CeuI wild-type values for the K_m and k_cat are 0.9 +- 0.3 nM and 3.7 * 10^-5 s^-1, respectively.

DISCUSSION

Using comparative sequence analysis and genetic selection, we have discerned a number of amino acid residues that are potentially important for the catalytic activity and DNA sequence specificity of the I-CeuI family of intron-encoded endonucleases. Genetic selection allowed us to constitute a collection of 49 I-CeuI variants at 37 distinct positions, the majority of which correspond to evolutionarily conserved sites. Our preliminary characterization of nine of the variants indicates that all of them behave differently from the wild-type enzyme with respect to the kinetics of the cleavage reaction. Three variants, each differing from the wild-type form by its charge, show undetectable endonuclease activity; five others feature reduced activity relative to the wild-type; whereas the remaining variant, located in the SNAT motif specific to the I-CeuI subfamily, differs from the wild-type in cleaving the top strand about three times more efficiently than the bottom strand. Taken together, our data provide a solid framework for future investigations on the structure/function relationships of I-CeuI and of closely related homing endonucleases.

At this time, we cannot describe precisely the specific residues that are directly involved in the function of I-CeuI (catalysis, recognition of DNA substrate, binding site for Mg²⁺) because several of the amino acid changes observed in the variants may have resulted in significant modification of the protein conformation, thus leading to changes in the enzyme activity. To demonstrate without any ambiguity which residues are functionally critical, analyses of enzyme kinetics and protein/DNA interaction using purified I-CeuI variants will be required in conjunction with site-directed mutagenesis experiments. Although such results are not available, a recent study on the yeast PI-SceI endonuclease by Gimble and Stephens (38 ) strongly suggests that the lack of I-CeuI activity in our E66K variant is due to the catalytic requirement of an acidic residue at the ninth position of the LAGLI-DADG dodecapeptide. Gimble and Stephens (38 ) have substituted the glutamic acid residue at this position in each of the two dodecapeptide motifs of PI-SceI with asparagine and alanine and found that both acidic residues are essential for catalysis. In future studies with I-CeuI, it will be interesting to test whether the evolutionarily conserved glutamine residue in the TQH motif participates in catalysis. No cleavage activity was detected in the variant showing a substitution with arginine at this highly conserved position among the various subfamilies of homologous proteins examined. The third I-CeuI variant we found to display no detectable cleavage activity (K116Q) exhibits a mutation in a residue specific to the subfamily of site-1923 endonucleases; we suspect that this residue is critical for substrate recognition.

The homing endonucleases containing a single copy of the LAGLI-DADG dodecapeptide may differ from PI-SceI in the structure of the active site. The results of Gimble and Stephens (38 ) support the idea that PI-SceI uses a single catalytic center to cleave both DNA strands of the substrate and that the conserved aspartic acid at the ninth position in each dodecapeptide is part of the catalytic center. The role of these two acidic residues is thought to be similar to the function of the conserved acidic residues that are found in the four restriction endonucleases whose structures are known (EcoRI, EcoRV, BamHI and PvuII). In these proteins, the acidic residues as well as a conserved lysine show almost identical positions with respect to the scissile phosphodiester bond (reviewed in 39 ). One model predicts that the acidic residues bind to the Mg²⁺ 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. A two-metal mechanism has also been proposed for EcoRV and BamHI in which each conserved acidic residue coordinates a Mg²⁺ ion (39 ,40 ). The present data provide no clues as to how I-CeuI and the homologous endonucleases containing a single dodecapeptide effect cleavage of their substrate. The alignment of I-CeuI and its homologues reveal no evolutionarily conserved acidic amino acid outside of the dodecapeptide which could replace the acidic amino acid in the second dodecapeptide. Assuming that two acidic amino acids are required to form the active site, then the second acidic residue might not be positionally conserved among the different protein subfamilies or each endonuclease might bind to the substrate as a dimer, allowing the formation of an active site comprising an aspartic acid from each dodecapeptide. Given that I-CeuI has been shown to be a monomer in solution (41 ), the latter possibility would imply the initial formation of a protein/DNA complex comprising one bound molecule of protein followed by the binding of a second molecule of protein. Alternatively, the active site of endonucleases possessing a single dodecapeptide may show little similarity with that of restriction enzymes and contains only one acidic amino acid. The conserved glutamine residue at position 93 whose replacement with an arginine abolishes I-CeuI endonuclease activity could be part of such a site.

Because a recent study indicated that only one of the two dodecapeptides in I-SceII (a dimer) is important for endonuclease function, it has been suggested that there might be fundamental differences between intron-encoded endonucleases and their homologues derived from inteins (42 ). The authors of this study found that substitutions of the conserved glycines at the fourth and tenth positions of the first dodecapeptide to aspartic acid block cleavage activity, whereas the equivalent substitutions in the second dodecapeptide inhibit maturase function. Drawing an analogy between I-CreI and I-SceII, the same authors noted that the dodecapeptide of I-CreI is likely to correspond to the first dodecapeptide of I-SceII and that this enzyme has no maturase activity, a function associated with the second dodecapeptide in I-SceII. The multiple protein alignment shown in Figure 4 strongly suggests that the second dodecapeptide of some LAGLI-DADG intron-encoded endonucleases could be essential for endonuclease function. All of the proteins containing a single dodecapeptide (including I-CreI, I-CeuI and I-CpaI) can be unambiguously aligned with the second half of their counterparts containing two copies of the dodecapeptide.

Several of the evolutionarily conserved positions in Figure 4 , in particular the positions specific to the I-CeuI subfamily, are not represented in our collection of I-CeuI variants. Because the sites of amino acid substitutions are clearly not randomly distributed and also because two or more amino acid substitutions are observed at nine sites of the protein, we believe that our failure to identify amino acid changes at numerous specific positions cannot be entirely attributed to an insufficient number of the mutant clones selected for analysis of the I-CeuI gene sequence. It is possible that single substitutions of a number of residues involved in DNA recognition are not sufficient to yield an enzyme without any toxic effect on E.coli. Some of the amino acid residues that are specific to the I-CeuI subfamily may also be important for a function other than endonucleolytic which is not selected against in E.coli. One such possibility is that of a maturase function, particularly given the inability of the CeLSU[middot]5 intron to splice in vitro (43 ), the remarkable stability of the endonuclease gene within the site-1923 LSU rDNA introns, and the larger number of conserved residues in the protein subfamily containing I-CeuI compared to those containing I-CpaI and I-CreI.

The E.coli cells carrying the five characterized I-CeuI variants with reduced cleavage activity can be propagated probably because the endonuclease activity is reduced to a level such that the target sites on the rDNA operons cannot be efficiently cleaved. We believe that the variant having the same K_m and k_cat values as the wild-type enzyme (T122A) is not lethal to E.coli because it more frequently cleaves the top strand. The single-strand breaks preferentially introduced by this variant can be religated readily by the E.coli repair system, whereas the double-strand breaks predominantly generated by the wild-type enzyme cannot.

Our inability to express the wild-type I-CeuI enzyme in E.coli contrasts with a previous report indicating that this enzyme was produced in E.coli (41 ). Another discrepancy between the results of this study and ours concerns the K_m of the wild-type enzyme. The value reported here for the in vitro-synthesized enzyme is 100-fold lower than the value reported for the recombinant enzyme produced in E.coli. In an attempt to resolve these discrepancies, we have sequenced the insertion of the expression plasmid which served to produce the recombinant enzyme and found a single mutation that causes the replacement of the arginine residue at position 156 (a residue specific to the I-CeuI subfamily) with a histidine. This mutation is probably responsible for the aforementioned discrepancies although we cannot eliminate the possibility that part of the reduced activity is due to the different kinds of substrate used. In addition, the presence of the His- and S-tags on the wild-type and variant enzymes studied here may alter the enzymatic properties of the endonuclease; however, such an effect proved to be negligible in recent assays with a purified variant enzyme (N.Drouin, L.D.Eltis and C.Lemieux, unpublished results). Production of the wild-type I-CeuI enzyme in E.coli would require a plasmid vector completely devoid of cryptic promoters or the expression of an inactive fusion enzyme which can be cleaved with a protease to recover the active form.

ACKNOWLEDGEMENTS

We thank Lindsay D.Eltis for critical reading of the manuscript. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to M.T. and C.L.) and from `Le Fonds pour la Formation de Chercheurs et l'Aide à la Recherche' (to M.T. and C.L.). M.T. and C.L. are Scholars in the Evolutionary Biology Program of the Canadian Institute for Advanced Research.

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*To whom correspondence should be addressed. Tel: +1 418 656 2131; Fax: +1 418 656 7176; Email: mturmel@rsvs.ulaval.ca
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