Catalytic properties of the HhaII restriction endonuclease

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.     

Chlorella virus DNA ligase: nick recognition and mutational analysis.

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.     

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme.nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.     

The reactions of the EcoRi and other restriction endonucleases.

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA--protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.     

Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRI. These single strands can be combined with their complementary single strands to obtain all possible EcoRI sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): (sequence; see text) The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRI was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRI. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRI than oligodeoxynucleotides containing EcoRI sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRI. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRI recognition site than for oligodeoxynucleotides containing an EcoRI site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRI. Our results allow details to be given for the probability of EcoRI making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occurring in vivo, since nicked intermediates can be repaired by DNA ligase.     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

MuA transposase separates DNA sequence recognition from catalysis

Confronted with thousands of potential DNA substrates, a site-specific enzyme must restrict itself to the correct DNA sequence. The MuA transposase protein performs site-specific DNA cleavage and joining reactions, resulting in DNA transposition-a specialized form of genetic recombination. To determine how sequence information is used to restrict transposition to the proper DNA sites, we performed kinetic analyses of transposition with DNA substrates containing either wild-type transposon sequences or sequences carrying mutations in specific DNA recognition modules. As expected, mutations near the DNA cleavage site reduce the rate of cleavage; the observed effect is about 10-fold. In contrast, mutations within the MuA recognition sequences do not directly affect the DNA cleavage or joining steps of transposition. It is well established that the recognition sequences are necessary for assembly of stable, multimeric MuA-DNA complexes, and we find that recognition site mutations severely reduce both the extent and the rate of this assembly process. Yet if the MuA-DNA complexes are preassembled, the first-order rate constants for both DNA cleavage and DNA strand transfer (the joining reaction) are unaffected by the mutations. Furthermore, most of the mutant DNA molecules that are cleaved also complete DNA strand transfer. We conclude that the sequence-specific contacts within the recognition sites contribute energetically to complex assembly, but not directly to catalysis. These results contrast with studies of more orthodox enzymes, such as EcoRI and some other type II restriction enzymes. We propose that the strategy employed by MuA may serve as an example for how recombinases and modular restriction enzymes solve the DNA specificity problem, in that they, too, may separate substrate recognition from catalysis.     

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.     

Sequential strand exchange by XerC and XerD during site-specific recombination at dif

Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.     

Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway

I-SceI is a homing endonuclease that specifically cleaves an 18-bp double-stranded DNA. I-SceI exhibits a strong preference for cleaving the bottom strand DNA. The published structure of I-SceI bound to an uncleaved DNA substrate provided a mechanism for bottom strand cleavage but not for top strand cleavage. To more fully elucidate the I-SceI catalytic mechanism, we determined the X-ray structures of I-SceI in complex with DNA substrates that are nicked in either the top or bottom strands. The structures resemble intermediates along the DNA cleavage reaction. In a structure containing a nick in the top strand, the spatial arrangement of metal ions is similar to that observed in the structure that contains uncleaved DNA, suggesting that cleavage of the bottom strand occurs by a common mechanism regardless of whether this strand is cleaved first or second. In the structure containing a nick in the bottom strand, a new metal binding site is present in the active site that cleaves the top strand. This new metal and a candidate nucleophilic water molecule are correctly positioned to cleave the top strand following bottom strand cleavage, providing a plausible mechanism for top strand cleavage.     

Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme

The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.     

Oligonucleotides with bistranded abasic sites interfere with substrate binding and catalysis by human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in oxidative damage DNA repair. The enzyme, which repairs abasic sites, makes a single nick 5' to the phosphodeoxyribose, leaving a free 3'-hydroxyl. We recently described single turnover kinetics for human recombinant AP endo acting on an oligonucleotide with a single abasic site. We hypothesized that the structural changes induced by the presence of a second abasic site might provide insight into how AP endo recognizes the first abasic site. Here we performed steady state and single turnover experiments using bistranded abasic site substrates, with the second site located on the complementary strand to the one being followed and either opposite to the first or displaced in the 5' direction. All sites on the complementary strand were within half a helical turn of the first. The catalytic efficiency was reduced 80 to 96% and the Kd for substrate binding and dissociation was elevated 40- to 125-fold. The smaller changes occurred when the second site was opposite the first site or displaced by four nucleotides. In addition, if the second abasic site was directly across the helix or displaced by 1 or 3 nucleotides from the first abasic site, cleavage of the first abasic site was subject to apparent substrate inhibition, which did not occur if the second abasic site was displaced by four nucleotides from the first. While a substrate containing a nick without a phosphodeoxyribose on the contralateral strand abasic site did not inhibit nicking of the first strand, a substrate with a nicked abasic site on the contralateral strand was an even stronger inhibitor of enzyme action than an oligonucleotide containing the corresponding abasic site on each strand. Consequently, the inhibitory effect of the second abasic site is probably the result of prior cleavage of the abasic site on the contralateral strand with resulting distortions to the DNA helix that interfere with enzyme binding and/or cleavage.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease

King et al. (King, K., Benkovic, S. J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 10(4) by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.     

Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease.

A complete understanding of the sequence-specific interaction between the EcoRI restriction endonuclease and its DNA substrate requires identification of all contacts between the enzyme and substrate, and evaluation of their significance. We have searched for possible contacts adjacent to the recognition site, GAATTC, by using a series of substrates with differing lengths of flanking sequence. Each substrate is a duplex of non-self-complementary oligodeoxyribonucleotides in which the recognition site is flanked by six base pairs on one side and from zero to three base pairs on the other. Steady-state kinetic values were determined for the cleavage of each strand of these duplexes. A series of substrates in which the length of flanking sequence was varied on both sides of the hexamer was also examined. The enzyme cleaved both strands of each of the substrates. Decreasing the flanking sequence to fewer than three base pairs on one side of the recognition site induced an asymmetry in the rates of cleavage of the two strands. The scissile bond nearest the shortening sequence was hydrolyzed with increasing rapidity as base pairs were successively removed. Taken together, the KM and kcat values obtained may be interpreted to indicate the relative importance of several likely enzyme-substrate contacts located outside the canonical hexameric recognition site.     

Crystal structures of the structure‐selective nuclease Mus81‐Eme1 bound to flap DNA substrates

The Mus81‐Eme1 complex is a structure‐selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross‐links, replication fork collapse, or double‐strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81‐Eme1, we determined crystal structures of human Mus81‐Eme1 bound to various flap DNA substrates. Mus81‐Eme1 undergoes gross substrate‐induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre‐ and post‐nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post‐nick DNA. These features are crucial for comprehensive protein‐DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81‐Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81‐Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends.     

Substrate dependence of the mechanism of EcoRI endonuclease.

The mechanism of EcoRI endonuclease is substrate dependent. At 37 degrees dissociation of the enzyme-Form II DNA intermediates of ColE1 DNA and bacteriophage G4 RFI DNA is negligible. Therefore, both DNA strands with in the EcoRI sequence are cleaved during a single binding event. However, double strand cleavage of SV40 DNA occurs without dissociation of the enzyme in only 75% of the catalytic events. This mechanistic difference presumably reflects sequence differences about the EcoRI sites of these DNA's.     

The SalGI restriction endonuclease. Mechanism of DNA cleavage.

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.     

Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein.

We previously demonstrated that the adeno-associated virus (AAV) Rep68 and Rep78 proteins are able to nick the AAV origin of DNA replication at the terminal resolution site (trs) in an ATP-dependent manner. Using four types of modified or mutant substrates, we now have investigated the substrate requirements of Rep68 in the trs endonuclease reaction. In the first kind of substrate, portions of the hairpinned AAV terminal repeat were deleted. Only deletions that retained virtually all of the small internal palindromes of the AAV terminal repeat were active in the endonuclease reaction. This result confirmed previous genetic and biochemical evidence that the secondary structure of the terminal repeat was an important feature for substrate recognition. In the second type of substrate, the trs was moved eight bases further away from the end of the genome. The mutant was nicked at a 50-fold-lower frequency relative to a wild-type origin, and the nick occurred at the correct trs sequence despite its new position. This finding indicated that the endonuclease reaction required a specific sequence at the trs in addition to the correct secondary structure. It also suggested that the minimum trs recognition sequence extended three bases from the cut site in the 3' direction. The third type of substrate harbored mismatched base pairs at the trs. The mismatch substrates contained a wild-type sequence on the strand normally cut but an incorrect sequence on the complementary strand. All of the mismatch mutants were capable of being nicked in the presence of ATP. However, there was substantial variation in the level of activity, suggesting that the sequence on the opposite strand may also be recognized during nicking. Analysis of the mismatch mutants also suggested that a single-stranded trs was a viable substrate for the enzyme. This interpretation was confirmed by analysis of the fourth type of substrate tested, which contained a single-stranded trs. This substrate was also cleaved efficiently by the enzyme provided that the correct strand was present in the substrate. In addition, the single-stranded substrate no longer required ATP as a cofactor for nicking. Finally, all of the substrates with mutant trss bound the Rep protein as efficiently as the wild-type did. This finding indicated that the sequence at the cut site was not involved in recognition of the terminal repeat for specific binding by the enzyme. We concluded that substrate recognition by the AAV Rep protein involves at least two and possibly as many as four features of the AAV terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease

Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu----Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.