Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding.

DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

Specificity of S-adenosyl-L-methionine in the inactivation and the labeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC, is irreversibly inactivated by its substrate AdoMet. AdoMet has two diastereomers with respect to its sulfonium center, (-)-AdoMet and (+)-AdoMet. We prepared (+)- and (-)-AdoMet from a commercial source, and compared their activities as a substrate and as an inactivator of ACC synthase isolated from tomato (Lycopersicon esculentum Mill). fruits. Only (-)-AdoMet produced ACC, whereas both (-)- and (+)-AdoMet inactivated ACC synthase; (+)-AdoMet inactivated the enzyme three times faster than (-)-AdoMet. We have previously shown that ACC synthase was specifically radiolabeled when the enzyme was incubated with S-adenosyl-L-[3,4-14C]methionine. The present results further indicate that S-adenosyl-L-[carboxyl-14C]methionine, but not S-adenosyl-L-[methyl-14C]methionine, radiolabeled the enzyme. These data suggest that the 2-aminobutyric acid portion of AdoMet is linked to ACC synthase during the autoinactivation process. A possible mechanism for ACC synthase inactivation by AdoMet is discussed.     

Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine

The chirality of biologically active S-adenosyl-L-methionine (AdoMet) is S,S, where the designations refer to the sulfur and the alpha-carbon, respectively. This paper describes a cation-exchange high-performance liquid chromatographic (HPLC) method for separating (S,S)-AdoMet from the biologically inactive (R,S)-AdoMet that results from racemization at the sulfur. This method was used to measure the rates of the degradation reactions of (S,S)-AdoMet as a function of pH. These reactions and the first-order rate constants, which were found at 37 degrees C and pH 7.5, are racemization, 1.8 X 10(-6) s-1; cleavage to homoserine lactone and 5'-(methylthio)adenosine, 4.6 X 10(-6) s-1; and hydrolysis to adenine and S-pentosylmethionine, 3 X 10(-6) s-1. Racemization showed no change in rate over the pH range from 7.5 to 1.5. The cleavage reaction persisted until the pH was lowered to 1.5, but hydrolysis ceased at pH 6. Commercial samples of nonradioactive AdoMet contained 20-30% (R,S)-AdoMet, while a sample of [methyl-3H]AdoMet had less than 1% (R,S)-AdoMet. Preparing enzyme substrates by mixing such samples will cause an underestimate of specific activity and an overestimate of the amount of product. The (R,S)-AdoMet/(S,S)-AdoMet ratio in mouse liver was 0.03, much less than the value of 0.19 calculated from the above rate constants. An enzyme extract from mouse liver did not degrade (R,S)-AdoMet, but a more thorough search may find such an activity. In any event, the cleavage and hydrolysis reactions partially balance the racemization of (S,S)-AdoMet in vivo and prevent excessive accumulation of (R,S)-AdoMet.     

Crosslinking of Dam methyltransferase with S-adenosyl-methionine

Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.

Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.     

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.     

Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates

Radioactivity from 3H-[methyl]-S-adenosyl-L-methionine (AdoMet) was covalently bound to protein-O-carboxylmethyltransferase and phenylethanolamine N-methyltransferase following 10-15 min irradiation by short-wave ultraviolet light. This photoaffinity binding of 3H-[methyl]-AdoMet was blocked by S-adenosylhomocysteine and sinefungin, but was not affected by 5 mM dithiothreitol. The binding was also inhibited by including methyl acceptors such as calmodulin (protein-O-carboxylmethyltransferase) or phenylethanolamine (phenylethanolamine N-methyltransferase) in the photoaffinity incubation. Staphlococcus V8 protease digests of 3H-[methyl]-AdoMet/enzyme complexes revealed that the primary structure around the AdoMet binding site is different in these two enzymes. Thus, protein-O-carboxylmethyltransferase, a large molecule methyltransferase, can covalently bind 3H-[methyl]-AdoMet in a manner similar to that of phenylethanolamine-N-methyltransferase.     

A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 microM (S,S)-AdoMet.     

S-adenosylmethionine: studies on chemical and enzymatic synthesis

Several methods for the chemical and enzymatic synthesis of (-)-S-adenosylmethionine (AdoMet) are described and compared. Studies on the effects of solvents, pH, methylating reagents, and KI on the coupling of sodium homocysteine thiolate and 5'-chloro-5'-deoxyadenosine led to an improved procedure for the synthesis of (+/-)-AdoMet. The use of trimethylsulfonium iodide as a methylating agent under acidic conditions results in a higher content of the desired (-)-epimer than does the use of CH3I. The enzymatic synthesis of (-)-AdoMet using AdoMet synthetase from an over-producing strain of Escherichia coli is demonstrated and the effect of product inhibition on preparative-scale synthesis is illustrated. A new HPLC technique for separation of the epimeric mixture of AdoMet, which allows small-scale preparation of optically pure AdoMet from the enzyme product, has been developed. With this HPLC technique, evidence that (-)-AdoMet is the sole epimer formed by the enzyme has been shown.     

ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids

Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.     

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.     

5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase

5-Fluorodeoxycytidine (FdCyd) was incorporated into a synthetic DNA polymer containing the GCGC recognition sequence of HhaI methylase to give a polymer with about 80% FdCyd. In the absence of AdoMet, poly(FdC-dG) bound competitively with respect to poly(dG-dC) (Ki = 3 nM). In the presence of AdoMet, the analogue caused a time-dependent, first-order (k = 0.05 min-1) inactivation of the enzyme. There is an ordered mechanism of binding in which enzyme first binds to poly(FdC-dG), then binds to AdoMet, and subsequently forms stable, inactive complexes. The complexes did not dissociate over the course of 3 days and were stable to heat (95 degrees C) in the presence of 1% SDS. Gel filtration of a complex formed with HhaI methylase, poly(FdC-dG), and [methyl-3H] AdoMet gave a peak of radioactivity eluting near the void volume. Digestion of the DNA in the complex resulted in a reduction of the molecular weight to the size of the methylase, and the radioactivity in this peak was shown to be associated with protein. These data indicate that the complexes contain covalently bound HhaI methylase, poly(FdC-dG), and methyl groups and that 5-fluorodeoxycytidine is a mechanism-based inactivator of the methylase. By analogy with other pyrimidine-modifying enzymes and recent studies on the mechanism of HhaI methylase (Wu & Santi, 1987), these results suggest that an enzyme nucleophile attacks FdCyd residues at C-6, activating the 5-position for one-carbon transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

A DNA-modification methylase from Bacillus stearothermophilus V.

A type II modification methylase (M BstVI) was partially purified from the thermophilic bacterium Bacillus stearothermophilus V. The methylase catalyses the transfer of methyl groups from S-adenosyl-L-methionine to unmodified double-stranded DNA. The product of methylation was identified by paper chromatography as N6-methyladenine. Since M BstVI protects DNA against cleavage by BstVI and XhoI restriction endonucleases, it follows that it methylates the adenine residue in the sequence 5'-C-T-C-G-A-G-3'.     

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.     

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

Cloning and characterization of the genes encoding the MspI restriction modification system.

The genes encoding the MspI restriction modification system, which recognizes the sequence 5' CCGG, have been cloned into pUC9. Selection was based on expression of the cloned methylase gene which renders plasmid DNA insensitive to MspI cleavage in vitro. Initially, an insert of 15 kb was obtained which, upon subcloning, yielded a 3 kb EcoRI to HindIII insert, carrying the genes for both the methylase and the restriction enzyme. This insert has been sequenced. Based upon the sequence, together with appropriate subclones, it is shown that the two genes are transcribed divergently with the methylase gene encoding a polypeptide of 418 amino acids, while the restriction enzyme is composed of 262 amino acids. Comparison of the sequence of the MspI methylase with other cytosine methylases shows a striking degree of similarity. Especially noteworthy is the high degree of similarity with the HhaI and EcoRII methylases.     

Isolation and Characterization of Biochemical Properties of DNA Methyltransferase <Emphasis Type="Italic">Fau</Emphasis>IA Modifying the Second Cytosine in the Nonpalindromic Sequence 5′-CCCGC-3′

A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.