DNA recognition of base analogue and chemically modified substrates by the TaqI restriction endonuclease.

It has been proposed that protein-DNA recognition is mediated via specific hydrogen bond, hydrophobic, and/or electrostatic interactions between the protein and DNA surfaces. We have attempted to map and quantitate the energies of these interactions for the TaqI endonuclease by constructing substrates substituted with base or phosphate analogues that either remove or sterically obstruct particular functional groups in the canonical TCGA sequence. The DNA backbone was also modified using a chemical approach (phosphate ethylation) which identified several phosphates in the recognition sequence essential for cleavage. The base analogues, N6-methyl-A, N7-deaza-A, N7-deaza-G, inosine, N4-methyl-C, 5-methyl-C, uracil, 5-bromo-U, and the phosphate analogues, alpha-thio-A, alpha-thio-G, alpha-thio-T, alpha-thio-A, were substituted for their corresponding unmodified counterpart in one strand of the TCGA duplex. The effects of these analogues were monitored by measuring the steady state (Km, kcat) and single-turnover (kst) kinetic constants. Only the N6-methyl-A-substituted DNA, which mimics in vivo methylation, was unreactive while the remaining analogue substitutions exhibited Michaelis-Menten kinetics. In general, the Km was either unchanged or lowered by the analogue substitutions. In contrast, many of the analogues severely reduced kcat, suggesting the modified functional groups served mainly to destabilize the transition state. Single-turnover measurements paralleled the kcat results, pointing to the N7 and N6 of A, the N7 of G, and one of the nonbridging oxygens 3' to T as putative contacts made in achieving the transition state. Substrates with double substitutions displayed simple additivity of delta delta G" implying that these changes behaved independently. The unmodified strand in 10 out of 12 hemisubstituted substrates had a normal kst value suggesting that a particular cleavage center is controlled predominantly by recognition of determinants on the same strand as the scissile bond. These results are discussed in relation to base analogue work from the EcoRI, RsrI, and EcoRV restriction endonucleases.     

Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase

A complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. IX. Cleavage of substrates with point modifications in the recognition site and flanking sequences

Ability of the EcoRII restriction endonuclease to cleave 14-base-pair DNA duplexes with nucleotide substitutions in the recognition site CCA/TGG and in the adjacent base pair has been studied. Modifications leading to a local change in the substrate conformation (rU residue in and outside the recognition site, A.A- or A.C-pairs in the flanking sequence) reduce the rate of hydrolysis, the effect being maximal when the modified base pair is outside the recognition site. No digestion occurs when the internal dC-residue of the recognition site is 5-methylated in one or both strands. Replacement of dT residue in the EcoRII recognition site by dfl5U residue results in a dramatic inhibition of hydrolysis. Km and kcat for the cleavage of 14-base-pair DNA duplex have been determined. The cleavage rate of the dT-containing strand of the recognition site in 1.5 fold higher comparing with the dA-containing strand. The cleavage of both strands of the substrate by EcoRII endonuclease is confirmed to proceed in one enzyme-substrate complex.     

Synthesis and properties of oligodeoxynucleotides containing the analogue 2'-deoxy-4'-thiothymidine.

The 2'-deoxythymidine analogue 2'-deoxy-4'-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oligoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2'-deoxy-4'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.     

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.     

Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.

A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.     

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.     

DNA recognition by the EcoRV restriction endonuclease probed using base analogues

The EcoRV restriction endonuclease recognises palindromic GATATC sequences and cuts between the central T and dA bases in a reaction that has an absolute requirement for a divalent metal ion, physiologically Mg(2+). Use has been made of base analogues, which delete hydrogen bonds between the protein and DNA (or hydrophobic interactions in the case of the 5-CH(3) group of thymine), to evaluate the roles of the outer two base-pairs (GATATC) in DNA recognition. Selectivity arises at both the binding steps leading to the formation of the enzyme-DNA-metal ion ternary complex (assayed by measuring the dissociation constant in the presence of the non-reactive metal Ca(2+)) and the catalytic step (evaluated using single-turnover hydrolysis in the presence of Mg(2+)), with each protein-DNA contact contributing to recognition. With the A:T base-pair, binding was reduced by the amount expected for the simple loss of a single contact; much more severe effects were observed with the G:C base-pair, suggesting additional conformational perturbation. Most of the modified bases lowered the rate of hydrolysis; furthermore, the presence of an analogue in one strand of the duplex diminished cutting at the second, unmodified strand, indicative of communication between DNA binding and the active site. The essential metal ion Mg(2+) plays a key role in mediating interactions between the DNA binding site and active centre and in many instances rescue of hydrolysis was seen with Mn(2+). It is suggested that contacts between the GATATC site are required for tight binding and for the correct assembly of metal ions and bound water at the catalytic site, functions important in providing acid/base catalysis and transition state stabilisation.     

Opposite base specificity in excision of pyrimidine ring-opened 1,N6-ethenoadenine by thymine glycol-DNA-glycosylases

A highly mutagenic DNA lesion, 1,N6-ethenoadenine ( epsilon A) is chemically unstable and either depurinates or converts to a pyrimidine ring-opened product upon water molecule addition to the C(2)z.sbnd;N(3) bond in epsilon dA (compound B). Compound B subsequently undergoes deformylation to yield compound C, which depurinates in the final step of the epsilon A rearrangement pathway. We have previously shown that epsilon A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which excises parental epsilon A. Compound B was shown to be eliminated from a B:T pair by Escherichia coli formamidopyrimidine-DNA-glycosylase (Fpg protein) and endonuclease III (Nth protein). Fpg protein excised B also from a B:C pair, and much less efficiently from B:A and B:G pairs [J. Biol. Chem. 276 (2001) 21821]. Here we show that efficiency of B excision by the Nth protein also depends on the opposite base in the pair. Most efficient repair is observed when this derivative is paired with dG (Km=18nM, kcat=12) and is less favourable when paired with dC (Km=40nM, kcat=13) and dT (Km=32nM, kcat=11). In physiological conditions, compound B is probably not excised by the Nth-glycosylase from a B:A pair, or from a single-stranded DNA, since kinetic constants in these conditions are an order or two orders of magnitude higher than when B is paired with T, C or G. A similar specificity for B excision was found for Saccharomyces cerevisiae Ntg2-glycosylase. Thus, when paired with A, an epsilon A derivative might be more persistent than when paired with other bases and give rise to AT-->TA transversions.     

Phosphonamidates as transition-state analogue inhibitors of thermolysin

Six phosphorus-containing peptide analogues of the form Cbz-NHCH2PO2--L-Leu-Y (Y = D-Ala, NH2, Gly, L-Phe, L-Ala, L-Leu) have been prepared and evaluated as inhibitors of thermolysin. The Ki values for these compounds range from 1.7 microM to 9.1 nM and correlate well with the Km/kcat values for the corresponding peptide substrates [Morihara, K., & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380] but not with the Km values alone. The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition-state analogues and not simply multisubstrate ground-state analogues. Cbz-NH2CH2PO2--L-Leu-L-Leu (Ki = 9.1 nM) is the most potent inhibitor yet reported for thermolysin.     

The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.     

Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence

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.     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.     

Purification and characterization of human 3-methyladenine-DNA glycosylase.

A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli. In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed. All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa. Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E. coli DNA repair proteins. All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA. The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable. In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein.     

The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence identically

Using a series of decadeoxyribonucleotides containing base analogues as substrates we measured the steady-state kinetic parameters for the reaction catalyzed by RsrI endonuclease and compared the results to those with its isoschizomer EcoRI. The kinetics of RsrI cleavage are affected by each substitution, with the effects being generally more deleterious than with EcoRI, as shown by the greater reduction in the specificity constant kcat/KM. The magnitudes of the effects of several substitutions are consistent with the formation of direct enzyme-nucleobase contacts at the indicated positions. With substrates containing 2-amino-purine or 2,6-diaminopurine at the central adenine or uracil at the outermost thymine in the recognition sequence, cleavage by RsrI was very slow, less than one-tenth the rate of the corresponding EcoRI-catalyzed reaction. The lower tolerance of RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms of DNA recognition by the two enzymes. Although RsrI and EcoRI endonucleases bind with similar affinities to specific and nonspecific DNA sequences and appear to introduce similar structural distortions in DNA upon binding, the use of substrate analogues reveals significant differences at the level of catalysis in the mechanisms by which these two endonucleases recognize the duplex sequence GAATTC.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.

The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-PorI. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-PorI, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-PorI cleaves optimally at 80 degrees C, with kcat and Km values of about 2 min-1 and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within a 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with those of eucaryotic homing endonucleases.     

Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease.

Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis.     

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.