Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein)

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

A study of penetrance of Drosophila melanogaster forked gene

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 A3/Da. The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.     

Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.     

FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex

The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.     

The non-catalytic function of XPG protein during dual incision in human nucleotide excision repair

XPG is a member of the FEN-1 structure-specific endonuclease family. It has 3'-junction cutting activity on bubble substrates and makes the 3'-incision in the human dual incision (excision nuclease) repair system. To investigate the precise role of XPG in nucleotide excision repair, we mutagenized two amino acid residues thought to be involved in DNA binding and catalysis, overproduced the mutant proteins using a baculovirus/insect cell system, and purified and characterized the mutant proteins. The mutation D77A had a modest effect on junction cutting and excision activity and gave rise to uncoupled 5'-incision by mammalian cell-free extracts. The D812A mutation completely abolished the junction cutting and 3'-incision activities of XPG, but the excision nuclease reconstituted with XPG (D812A) carried out normal 5'-incision at the 23rd-24th phosphodiester bonds 5' to a (6-4) photoproduct without producing any 3'-incision. It is concluded that Asp-812 is an active site residue of XPG and that in addition to making the 3'-incision, the physical presence of XPG in the protein-DNA complex is required non-catalytically for subsequent 5'-incision by XPF-ERCC1.     

I-NjaI, a nuclear intron-encoded homing endonuclease from Naegleria, generates a pentanucleotide 3' cleavage-overhang within a 19 base-pair partially symmetric DNA recognition site

Different species of the amoebo-flagellate Naegleria harbor optional group I introns in the nuclear ribosomal DNA that contain open reading frames. Intron proteins from Naegleria jamiesoni, Naegleria andersoni, and Naegleria italica (named I-NjaI, I-NanI and I-NitI, respectively) were expressed in Escherichia coli and found to be isoschizomeric homing endonucleases that specifically recognize and cleave intron-lacking homologous alleles of ribosomal DNA. The I-NjaI endonuclease was affinity purified, characterized in more detail, and found to generate five-nucleotide 3' staggered ends at the intron insertion site which differs from the ends generated by all other known homing endonucleases. The recognition site was delimited and found to cover an approximately 19 base-pair partially symmetric sequence spanning both the cleavage site and the intron insertion site. The palindromic feature was supported by mutational analysis of the target DNA. All single-site substitutions within the recognition sequence were cleaved by the purified I-NjaI endonuclease, but at different efficiencies. The center of symmetry and cleavage was found to be completely degenerate in specificity, which resembles that of the subclass IIW bacterial restriction enzymes.     

Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations

Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a [2Fe-2S] ferredoxin. Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953, 207-213). We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding. Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c. Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively). In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively). Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins. These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.     

The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.     

Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase

Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD+-specific isocitrate dehydrogenase (IDH). The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding. The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP. In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in Vmax due to a 70-fold increase in the S0.5 value for NAD+. These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands. In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects. A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.     

The pH-induced release of iron from transferrin investigated with a continuum electrostatic model

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pKa values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue's pKa value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H+ ions. It is suggested that the release of these three H+ ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.     

Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter

The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Cysteine-scanning mutagenesis around transmembrane segment III of Tn10-encoded metal-tetracycline/H+ antiporter

Each amino acid in the putative transmembrane helix III and its flanking regions (from Gly-62 to Tyr-98) of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) was individually replaced with Cys. Out of these 37 cysteine-scanning mutants, the mutants from G62C to R70C and from S92C to Y98C showed high or intermediate reactivity with [14C]N-ethylmaleimide (NEM) except for the M64C mutant. On the other hand, the mutants from R71C to S91C showed almost no reactivity with NEM except for the P72C mutant. These results confirm that the transmembrane helix III is composed of 21 residues from Arg-71 to Ser-91. The majority of Cys replacement mutants retained high or moderate tetracycline transport activity. Cys replacements for Gly-62, Asp-66, Ser-77, Gly-80, and Asp-84 resulted in almost inactive Tet(B) (less than 3% of the wild-type activity). The Arg-70 --> Cys mutant retained very low activity due to a mercaptide between Co2+ and a SH group (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Three of these six important residues (Ser-77, Gly-80, and Asp-84) are located in the transmembrane helix III and one (Arg-70) is located in the flanking region. These four functionally important residues are located on one side of the helical wheel. Only two of the residual 31 Cys mutants were inactivated by NEM (S65C and L97C). Ser-65 and Leu-97 are located on the cytoplasmic and periplasmic loops, respectively, in the topology of Tet(B). The degree of inactivation of these Cys mutants with SH reagents was dependent on the volume of substituents. In the presence of tetracycline, the reactivity of the S65C mutant with NEM was significantly increased, in contrast, the reactivity of L97C was greatly reduced, indicating that the cytoplasmic and periplasmic loop regions undergo substrate-induced conformational change in the mutually opposite direction.     

Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.     

Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p1), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p2 and p0 have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3' side and 5' side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p2, and Lys-66 in p0) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p2 are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p0 electrostatic interaction contributes only to binding of the RNA.     

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 A resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.