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CHAPTER-II: TYPE II RESTRICTION ENDONUCLEASES AND LITERA'l'URE REVIEW ON BamHI: ================================================================================ A] RESTRICTION AND MODIFICATION OF DNA 28 B] TYPE II RESTRICTION ENDONUCLEASES 28 (i) (ii) (iii) C] General Properties Structure Of Restriction Endonucleases (a) EcoR I (b) EcoR V Catalytic Mechanism Literature Review on BamH I 37 (A) RESTRICTION AND MODIFICATION OF DNA Restriction identified involved 1968). and modification of DNA in bacteria were first over 30 years ago and the first characterization of an in this process was described 25 years ago (Meselson A R/M system must possess two enzyme activities, the enzyme and Yuan restriction endonuclease and the modification methylase, both of which are dependent on recognition of 1982) same The restriction sequence so the DNA sequence (Bickle 1982; Modrich activity cleaves the DNA, but only if is not methylated. there is no cleavage barriers Arber Roberts recognition In the bacterium both system are active of its own genome. But this mechanism and provides against both interspecies genetic transfer and phage infection 1979). Many R/M genetic analysis reveals three system have now been characterized and subsequently different 1987). For type I and classes III of type I and two in the i.e., type trast the type II systems consist fication and one for restriction of type I, II and III by (Bickle are functions of one of different case initially by purification of the proteins and it both activities meric protein made up of a number case and oligo- polypeptides, three in the III (Bickle 1982). In of two separate proteins, one for conmodi- (Modrich and Roberts 1982). \B) TYPE II RESTRICTION ENDONUCLEASES (i) General properties Type II restriction endonucleases are the simplest and the common (Roberts 1990). teins. prise separate The recognition sequences are essentially symmetric, Cleavage in The to act mainly as homodimers, the R and M genes occur in all linkage 28 The com- additional interruptions (Wilson occurs symmetrically within the sequences. believed monomers. the form of non specific pro- they four to eight specific nucleotides, but they may include nucleotides are The endonuclease and methylase are most 1991). endonucleases methyltransferases configuration as (Wilson 1988). Most first; at other genes often, the genes are aligned; sometimes the R genes times the comes M genes come first. In several systems have opposite orientations; some diverge, other converge the (Wilson 1991). A tremendous amount of diversity is seen II restriction enzymes. among the various Type At present over 640 different type II restriction enzymes have been identified from a wide variety of bacterial genera between them, 1987). The Nathans nomenclature given by (Roberts Smith the same DNA sequence. which each usually Most position 1987). Hha I, EcoRI tion sequence. observation But more specific for (pu) which the specified Moreover, DNA sequences. and pyrimidines (py). for Cau I and and these in are recogni- For example, Hind II sequence sequences, EcoRV provide examples of this type of bases. unique to (Roberts quences in which certain position tides. either other enzymes such as Hha II and unspecified found 4 or 6 bp or rarely 8bp discontinuous sequences in one or unique is fully-specified by a given base, and of Two or more of such enzymes are known as of these enzymes recognize continuous sequences of and The discrepancy between the numbers enzymes and recognition sequences arises from the "isoschizomers". Cau sequences restriction enzymes from different bacteria are frequently recognize are DNA widely accepted is that (Smith and Nathans 1973). restriction that they recognize over 135 different and, Sfi recognize bases- are interrupted by not all restriction enzymes Many recognize can be I, degenerate se- occupied by alternative nucleo- can act upon various combinations of purines Second type is exemplified by the Cau II. recognition The central position of DNA sequence I is either A-T or T-A while for Cau II its target sequence can have either G-C or C-G as the central Unique recognition sites for restriction enzymes are nearly always symmet- rical, both strands of the sequences are number type II quences of called base pair. for DNA having palindromes. restriction (Bennett and Halford; 1989.) the same However, enzymes that but at sites that have no symmetry 29 5'-3' there also exist a recognize at all; sequence. Mba unique I Such small DNA se- provides one such example. Degenerate rical or asymmetrical, either recognition sequences depending on I or Hind II). both strands of DNA within be either by the degeneracy retain The majority of type II restriction their symmet- the nature of the degeneracy, but in case the nucleotides unaffected (Cau may recognition sequence With some, the cleavage occurs in the centre of symmetry enzymes cleave (Roberts 1987). the recognition site (for example, EcoR V or Hind II), and in these cases the reaction products will be double stranded DNA with flush termini. cleaved and towards the 5' end of the With others, recognition sequence Eco RI) or towards the 3' end (viz Hha I), products will be duplex DNA that carry single 5' or 3' termini, respectively. both strands are (example Cau and here their I reaction stranded extensions at their However, when two or more restriction enzymes cleave DNA at the same site, these isoschizomers do not necessarily cleave the same phosphodiester bonds. For example, the DNA recognized by Cau II is also the target for both Nci I and while I cuts the same bonds as Cau II, Ben I cleaves the Nci different position generalization their the that (Roberts 1987). There are is type II restriction enzymes cleave the reminiscent (Bickle 1987). belong to the none cleave DNA but, at to DNA of type I and type III type II category. restriction First they require Mg++ as the within In some ways, However, by two criteria 'enzymes such as Mbo a cut the enzymes II clearly a cofactor of the other cofactor needed for type I and III and second, they the recognition zymes I, excepti{)ns DNA at some distance from their recognition sites. but Ben recognition sequences. Several of these enzymes, such as Mbo II behaviour DNA at fixed rather then variable sequence (Brown et al., 1980). hydrolyse droxyl groups. phosphodiester The bonds different aspects of small number of distances All type II away from restriction to leave 5' phosphate and 3' enhy- type. II enzymes are thus a diverse and heterogeneous group of proteins and individual enzymes very also sequence can be exploited to analyse DNA-Protein interactions. However, at present only a restriction enzymes have been analysed in -any chemical detail and in terms of their recognition to X-ray crystal structure is some extent EcoR V ; their 30 many except for EcoR I known bioand though anything har~ly about the cleavage mechanism is known. (ii) Structure Cocrystal structure of two restriction enzymes ECoR I and ECoR with their cognate sequence bound in absence of Mg++ have been V analysed in great detail. (a) EcoR I : Of best the characterized symmetric many is the EcoR I system. that homodimer The EcoR I endonuclease dimension 1989; 1984; and in the DNA and enzyme may occur McClarin ·et structures studied by studies al., of 1986; Kim naked these finding (Lane et al., 1991) anomalies at EcoRI sites, the more that bends it to substrate flanking DNA et the a structure in has and been are bound or EcoRI 50° angle (Kim et a unwinds sites 1991). with analysis, More the recent structural increased flexibility conformation EcoRI backbone flanking al., 1984) .. In the revised the original the structure and present have disappeared (Frederick et al., 1984; McClarin et al., 1990; Rosenberg 1991). Current model 31 EcoRI suggests site, in the 25° EcoRI- al., 1986; that is sites DNA by approximately central kink is present in kinks observed in al., EcoRI sites have been containing the phosphate in et Rosenberg controversial. kinks al., DNA-protein elestrophoretic consistent one Conformational specific 1990; oligonucleotides kinks are present while a recognition 1984; Frederick al., protein either showing B-DNA. 3lp NMR of naked EcoR I sites and in flanking backbone. However overall DNA backbone like confirms and et X-ray crystallography, NMR interpretation of in Mclanghlin 1988). to trigger DNA scission (Kim et. al., Although NMR scans by recognizing distorted B-form DNA (Nerdal et et al., 1991; Dickmann complex is In vitro studies revealed EcoR (Jacket al., 1982; Terry et al., 1985) to find its Lane changes endonuclease initially binds non-specifically and then site GAATTC, possibly the cleaves the sequence GAATTC between G and A on both DNA strands (Bennet and Halford 1989). I systems known restriction-modification but naked Kim kinks flanking EcoRI sites serve Following produce features of and distorts DNA flanking the naked the original structure, such as the DNA site. binding However, other aspects differ; the amino is widened by 3.5A 0 to permit access the increased buckle, site, central thymine carboxy- the major Each complex, and an groove,- Several the proposed al., 1986; al., 1989; and GAATTC adenines the and and anchors DNA- centre the the scissile bonds in the chain that steeply penetrate and of DNA, active by traverse laboratories when tested the original model by contact residues like glu144 arg145 and et arg200 al., 1989; Heitman and Model 1990; Oelgeschlager et al., 1990; Osuna et al., Conservative substitutions (arg200 __ lys, substrate specificity, (Wolfes et al., 1986). at retained considerable DNA mutations and respectively. substitutions hydrogen rna- residues that recognizes 'GAATTC' are borne possible in major the monomer bears an arm that enwraps extended Needles inconsistent. Heitman of 2 fold axis of symmetry through the each subunit, two ex-helices alter enzyme is a symmetric dimmer a sites. the In I shows I EcoR sites. DNA angles roll the backbone is kinked at the centre EcoR complex stabilizes The al., recognition the unusual et carbonyl oxygen alter the inner AT basepair conformations. The the pairs have Some ex-helices, and the central hydrogen bonds between the central and enzyme base to to residues, and the The DNA structure in the complex is quite unusual. chinery, ity bends, by EcoRI. II neokinks flanking the EcoR I site have disappeared (Kim groove not unwinds, recognised have moved, the arm is now composed of internal 1990). the initial signspot central kink and undo kinks are still present. type an specific binding EcoR I a termini as - glu144, arg145 and arg200, cleavage activity and Model 1990). None were altered vivo or in vitro, bond the affecting even in though some DNA site, in addition, these three residues 32 altering (Wolfes et Alves et 1991) found glu144 __ gln) Among 50 several (Needles et it did to 60 mutants al., 1989; their substrate. specificsubtitutions while severely double impaired could and not triple catalysis, they did not alter chlager et al., 1990; The roles, rather revised crystal structure (Fig. 8a,8b) reveals two residues to glu144 is near DNA it is not bound to the different adenines but and hydrogen bonds with other residues. Asn141 to bidentate hydrogen bond the adenines (GAATTC). Arg200 was thought to hydrogen bond guanine, now cooperates with arg203 which chelate a water molecule that binds guanine (Rosenberg 1991). This is consistent with the finding matic activity but that arg200 (Yanofsky by (Fig. 9). Substituting to model enzy1990). lys (Heitman 1989) or gluta- et al.,1987) inactivates EcoR I. arg145 does bidentate hydrogen bond, tions substitution decreases not substrate specificity (Heitman and Model Arg 20 3 is essential because substitution mate to contact DNA glul44 and arg200, fulfill salt bridges is now proposed This lead of the crystal structure. proposed forms Oelges- al., 1989; revised while Osuna et al., 1991). et a detailed analysis originally substrate recognition (Alves As originally proposed, span, and recognize adenine N7 posi- arg 145 by lysine or cysteine does not alter substrate specificity (Heitman and Model 1990), most probably because contacts to adenine N6 amino groups and thymidine suffice to discriminate substrate. A novel DNA recognition element which has so not far been found in other DNA binding proteins, an extended chain composed of residues 137 to 142 contacts amino lies within the DNA major groove and makes van der with pyrimidines and hydrogen bonds with pyrimidines and groups. Within this extended chain lies ala138. adenine alal38 main chain keto oxygen qydrogen bonds with the cytosine N4 amino group asn 141 hydrogen bonds to two adenines N6 amino groups while met 137 The cytosine-5-position, gly140 with the outer thymidine The Waals methyl ala142 with the inner thymidine methyl group interact by van forces (Rosenberg 1991).· In addition to the pyrimidine contact group der with and Waals from the extended chain, van der Waals interactions are proposed between ilel97 and the cytosine 5-position, and glnllS and the inner thymidine methyl group. 33 Figure , . Schematic backbone drawing of one subunit of (dimeric) £coR I endonuclease and both strands of the .DNA in the complex. The arrows represent~ str&nds, the coils represent o helices and the. ribbons re~resent the DNA backbone. The helices in the foreground of the diagram are the inner and outter recognition helices. They connect the third 8 strand to the fourth and the fourth ~ stund to the fifth. The two helices also for~~~ the centrAl interface with the other subunit. The amino·terminus of the polyper-tide chain is in the arm near the DNA. FIG- 9 (b) EcoR V : its Recently cognate shows DNA EcoR (Winkler et al., 1991). V to be a structural similarity the of lack Furthe rrno re, tortions DNA amino acid appears loop catalysis Therefore, I-DNA acidic of with the loops. enzyme However no is found as expected ~coRI similarity in unbound would In bonds may between both from protein. since EcoR and bound I form the distortions complex with with bases in of the and EcoR cognate major the ++ bindi,ng scissile bond. V are with DNA, disquite are participate in Mg the vicinity be quite distinct between (iii) catalytic Mechanism Analysis in that The DNA complex. residues which are located both recognition sequence more like A-DNA. it appears unrelated EcoRV and specific hydrogen makes groove and three and between from the EcoR a The structure protein with protruding ~~~ elucidated the structure of the DNA in EcoRV-DNA cocrystals shows and different the structure of EcoR V has been structurally mechanism for DNA the two enzymes. ! of the mechanism of action of restriction enzymes have two types of DNA substrates either long DNA molecules of length 1-50 kilobase pairs or synthetic oligonucleotides usually between 8 and 12 base used pair long rial plasmids, or phage or viral genomes fully sequenced, have cessfully (Greene et al., 1975). used. covalently of closed cutting very at been a copy product formed by cutting one site remains a substrate for other sites, and different sites on the same kinetics circle (ocDNA) cut the (Halford and Johnson 1980). DNA The can show reason the is cccDNA is that it reveals directly the mode recognition open This simplifies the kinetic suc- been circle of duplex DNA (ccc DNA ) that contains one by the restriction at the bacte- for substrate cleavage of analysis, different ideal strand the range For mechanistic studies the ideal substrate has the recognition sequence. otherwise For large DNA a wide enzymes. of DNA The enzyme initially cuts just one site on the 'ccc' substrates to generate form. A subsequent reaction will then be required second strand of the recognition site, thus converting 34 the the to oc form of the DNA vert the ccc detected. to the Alternatively, the enzyme may three forms electrophoresis of DNA can be separated from each through agarose. mixture at time intervals, and stopping for cutting each strand can be belled DNA or densitometry of this very turnover and transient kinetic amounts type to parameters jian the for 1987; for normal The for and these of for enzymes. enzyme, Moreover, and these the values of kcat and act as a competitive necessary nature of cannot be compared the the Halford 1982). Type II pairs at its recognition site, pairs. In addition, on DNA DNA can restriction kro values for their recognition sites sequence specificity. kro is that for Hha II; between recognition site This is not to the that the 4 base for the biological function but also is intrinsic DNA values temperature kro measured the state However, substantially· DNA typically of the order of 1 nM. highest more base the reaction at and generally display low plasmids or phage only to et al.,l980; Maxwell endonuclease on inhibitor with Chirik- 1976). macromolecule will always be apparent values, as the rest of (Langowski and For each enzyme differ turnover steady are determined under reaction condition and that time. format the enzyme 1983; cataly- catalytic Modrich and Zabel with those for the other enzymes. suitable single measures of enzyme Michaelis-Menten reasons the constants for each ~ almost given several of these enzymes are known (Nardone directly kcat As both at a DNA concentration, and value for Imber and Bickle 1981; various to behaviour kinetics II enzymes follow the standard respect time radiola- This assay simply of ccc, oc, and linear forms of the DNA (Modrich and Roberts 1982). by the experiments (Halford and Johnson, Type II restriction enzymes show the sis either by number (Kcat < 10 min-I minor modification Terry et al., 1985; Halford and Goodell 1988). the other reaction, stained gels. low turnover method can be adapted with the obtained by using ethediurn bromide all restriction enzymes have a being Hence, by removing aliquots from course ), con- substrate directly to linear DNA without any oc forms The reaction linear form. It may this enzyme where as all be significant must interact with others interact with 5 or restriction enzymes 35 have much lower Kcat values, values typically 1 min-I than the majority of where enzymes of above 100 sec-1 are commonplace (Fersht 1985). Given the similarities in the steady state parameters for different restriction enzymes, it might be anticipated that all of these use the same mechanism to cleave the DNA at their sites, this is not so (Halford 1983). At least two recognition basic mechanism been described for different restriction enzymes:either the have of but respective enzymes each cleavage strand of the DNA take place by individual reaction that separated kinetically, or the cleavage of both strands by a can be concerted reaction in which the cutting of one strand cannot be separated kinetically from that of the other strand. However, the categorization of a restriction enzyme to one mechanism or another is essentially arbitrary. A restriction enzyme can cleave one DNA substrate by one mechanism and another substrate by different the other, or alternatively it may cleave one substrate mechanisms under different by reaction conditions ( Maxwell and Halford 1982; Halford and Goodall 1988). The first use of- a synthetic oligonucleotide as a substrate for This study three principal characteristics of these substrates. These the synthesis of a self-complementary oligonucleotide that contains the recognition sequence, so that it can anneal to itself to form for the and this substrate gave a non-linear Lineweaver-Burk plot a demonstrated were, a enzyme restriction first, duplex. Reduced oligonucleotide was by Greene activity was et al., observed (1975). above the Tm until corrected for the fraction of single-stranded material. value enzyme of Kcat was for cleavage of this oligonucleotide same as that for the DNA by macromolecule. the Second, restriction However, another oligonucleotide substrate for the same enzyme with different flanking p~irs, gave a much higher value of Kcat the (Brennan et al., base 1986). Oligonucleotides may well be cleaved by restriction enzymes faster than DNA macromolecules, steps: an for, these enzymes may dissociate from the latter initial transfer of the enzyme from the 36 recognition in two site to nonspecific DNA, which then retains the enzyme until the final dissociation (Terry et al., 1985). Obviously facilitated diffusion can play no part in the reaction with oligonucleotides. (C) LITERATURE REVIEW ON Bam HI: The type II restriction endonuclease Bam HI from Bacillus liquefaciens 3' recognises (Wilson and subject of the pressed,. its Many the symmetrical sequence Young 1975; Roberts et al., 1977). pr~sent X-ray biochemical duplex 5'-GGATCC- This enzyme, thesis, has been cloned,sequenced crystal structure has still not and kinetic data are, however amylo- and been the overex- elucidated. available as discussed below. The restriction and specific endonuclease modification system in bacillus I is a part of amyloliquifaciens by hydrolysis of the phosphodiester bonds fashion across the hexanucleotide termini (Wilson and Young a cofactor (Roberts et sequence 1975). al., in generating the H (Wilson Young 1976; Shibata and Audo 1976; Shibata et al.,l976). DNA stricted as BamH a is staggered 5'-phosphoryl For catalysisy Bam HI requires 1977). The mechanism re- of cleavage Hg++ can be explained as follows (E+E).(S+S) --> (E+E).(S+P) --> (E+E) (P+P). Here E+E is the dimeric enzyme and S+S is the duplex DNA that is cut in one or both the transient both strands to yield S+P and P+P, respectively. existence means that the dissociation of the product cut strands (P+P) must be the slowest step in the pathway, and this determine the transient phase the phase The steady-state means rate. The absence of oc DNA (S+P) from that the formation of this intermediate first strand) is much slower than its breakdown (cutting . 37 of in must the (cutting the second - strand). Gel filtration of Bam HI endonuclease at ionic strengths 0.3 M NaCl and lar weight protein concentration of O.lmg/ml shows of 90,000. was estimated ugation. a native In the presence of 0.5 m NaCl the molecular SDS-PAGE revealed a single band with weight centrif- weight molecular of These suggests that the endonuclease consists of identical polypeptide chains which aggregate to dimerise or tetramerise depending strength. ionic th~ to molecu- to be 46,000 by gel filtration and sucrose density 22,000 daltons. up range, with 1OrnM. The addition Bam HI endonuclease exhibit activity over optimuin at 8.5 in tris buffers. an The on broad pH Mg++ is optimum Tween 20 or BSA to the reaction solution greatly activity at 37°C. The enzyme is most active at 37-40°C and appears to be stable to thermal denaturation upto 45°C in the absence of substrate in stabilizes presence enzyme of or of Nacl. However, the enzyme is lOOml Nacl. salt 10-50 mM lower It can be further stablized to 65°C optimally the active and is severely inhibited at 250 mM Nacl. DNA at also has a stabilizing effect at 37°C when protein solutions are dilute (Nardone and Chirikjian 1987). The _purified buffer Mg ++ at Mn++ zn++ with loss cu++ could performed with form site) substitute for Mg++. not estimated turnover shown to al., data subsets be containing GG studies The 5'-phosphoryl (Lee and Chirikjian HI nm. deoxydi- Bam HI sequence (GG,GA,AT,TC,CC) have inhibitors et 1979; been George al., 1980) reciprocal plots of representative and CC indicated unrelated ·could not inhibit varied the specific Hinsch Initial velocity with Kms in the vicinity of 3.6 number was 1.5 min-I. of 1985; Dinucleotides kinetics re- replaced Sv40 or pBR-322 DNA (both containing one Bam I in Other divalent cations like ca+ ~ of 80% of activity. revealed hyperbolic nucleotide et year optimal concentration of lOmM which may be an and The one containing 50% glycerol (Smith and Chirikjian 1979).The enzyme quires by enzyme is quite stable for at least to the Bam the endonuclease. over wide range suggesting competitive HI sequence The inhibition such as AA,TT, Ki values patterns. and of the dinucleotides differences in the interactions of 38 GC the endonuclease at various points within the dinucleotides CC and GG had the ends site are of the recognition The inhibiting effects of most recognition potent most Kis, suggesting important the dinucleotides were found changes in the enzyme caused the binding of another. for the the inhibitors. The the symmetrical to conformational meas- SV40 DNA complex (formed >1 suggesting multiple construct Hill interacting existence of at least two binding active site for each inhibitor to to be synergistic the dinucleotide were used to Hill coefficients were activity. filter binding experiments stable endonuclease by Mg++) the binding of an inhibitor which affects Nitrocellulose uring the displacement of plots. by the The that for enzyme in mixing experiments. This has been presumed to-be due in the absence of sequence. sites sites in has been taken into consideration due double stranded nature of BamH I site (Nardone and Chirkijian 1987). The kinetics o(, Bam HI endonuclease was examined with full length pBR322 DNA that had been linearized with different restriction enzymes. The endonuclease exhibited faster reaction rates with substrates having the recognition site Experiments that with or 5') of position (Nardone et Km the If or target the nearest end), it is This would be for ascertain Bam HI, enzymes direction DNA a a and 1921 presumed that more types of facilitated can locate their recognition site by then an enhancement of these process manifestation non-specific binding if facilitated centrally located the Bam HI a would central of the proportionately around a compared occur as the recognition site is located in a more area to these hoping mechanism be expected to position. from showed There was (Bam HI Cleavage site is 375 rate differences originate from one or diffusion. sliding for EcoRI linearized pBR322 pBR 322 DNA. pairs, respectively, reaction order in Ndei linearized base al.1986). a variety of linearized pBR 322 DNA substrates the nearest DNA terminus to the Bam HI site. fold increase to the a more central these effects were connected with the distance and not the (3' 3 in longer site. In diffusion is kinetically evident for Bam HI site on linearized pBR 322 was 39 used. It was 74 observed bps to 4362 bps that when the cleavage rates increases folds. cleavage In absence of NaCl the kinetic preference for longer observed that greatly reduced by 9 between the rates substrate length is increased Differences in the presence of 160 mM was rate limiting the increases in Nacl. substrates over a DNA concentration range of O.l-12nm. With association from the the was assumption cleavage rates could have been due to the longer target area for non-specific binding and, therefore, an increase in the frequency of facilitated transfer. The elimination rates of both between consistent cific differences the full length substrates by high with a decrease in the cleavage the I activity. of of non second, Ba~ This secondary activity called activity of the same enzyme but is induced by Bam HI HI.l has been seen to be altered in the presence of hydrophobic from the Bam HI palindrome. High alkaline conditions the enzyme uifaciens (George et al., 1980). strains strains 1976 F apart However, various from strain H have ), while strain N showed two activities called Bam NI and Bam Nx (Shibata, having same the molecular discrete The Mg++ weight bands specificity as Bam HI, than Bam as compared to concentration Nx the and was distinct to cleaved phage latter which gave requirements for optimum 40 to Bam to amongst (Shibata et endonuclease and Audo 1976 ). found served B.amyloliq- found and · K showed same cleavage specificity in- inherent other .been were and But the conversion of Bam HI is found to be reversible and therefore the property is which DNA enzyme concentration in the presence of organic solvents optimize the secondary activity. the The specificity of cleavage different creased of to or the activity such as glycerol and DMSO. Cleavage sites in 0 174 RF al., spe- was found reagents, HI.1 is distinct alteration It is also termed as "Star activity" observed due to relaxed specificity of Bam HI. to cleavage concentration electrostatic component seems to have a trace- amount reaction conditions. of NaCl in binding. BamH be long chain preference and the have A BamN a lower into five fifteen activity I were bands. found to different, also, phage 105-C.H be by protected showing Barn NI of while Ban Nx gave eight bands instead fifteen the degeneracy of the reaction. Amino various acid residues required for activity have been biochemical sensitivity and completely grown on strain H was methods. Bam HI endonuclease was studied found to by have towards reagents that modify sulphydryl groups. At neutral pH 37°C, incubation of the enzyme with 6mM ioodoacetamide, dithiobis (2- nitro benzoic acid) or N-ethylmalemide for 45 minutes resulted in 45%, 23% and 30% inhibition of activity respectively. Using 5,5'-bis (2-nitobenzoic acid) DTNB and p-mercuribenzoate it has been shown that Bam HI lost its activity (Wells, et al., 1981). endonuclease that contain the nucleotide or phosphate-binding sites (George an which were nition arginine specific reagent, inhibited presence preliminary of sequence. inactivation. recognition These studies enzymes role of these amino al.,1985) by inhibitors HI palindrome. acids in enzyme most Dinucleotides that were failed to protect the enzyme ~hat arginine residues may the from reside sequence-specific Different variants of Bam HI that cleavage activities were found to have cl~ave catalysis. The active site has been shown to charged pocket comprising largely of Aspartate coordination complex with divalent Mg++ ions the process of catalysis. pyridoxal and during The loss of these moieties allows the enzyme to (Shuang Yang lysyl residue also can with indicate the and aspartate residues indicating direct or indirect negatively bind but not to sequence undetectable Glutamate which forms residues proteins decreased competitive active site and might function in mutation at glutamate of HI endonuclease was dinucleotide pdGpdG protected the butanedione modification. displayed reduced or a et the The inhibition of the enzyme with DNA or The recognition of the Bam have Bam the 5'-phosphoryl deoxydinucleotide subset of the Bam HI recog- unrelated to the the sodium borate. incubation efficiently against in in have been examined because of their alleged role in Butanedione, in Arginyl residues completely not Xu be ruled and Ira Schildkraut 1991). Role out since modification of lysyl phosphate inhibit Bam HI and Chirikjian 1987). 41 activity (Nardone