Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

Determination of the recognition sequence of Mycobacterium smegmatis topoisomerase I on mycobacterial genomic sequences

Mycobacterium smegmatis topoisomerase I has several distinctive features. The absence of the zinc finger motif found in other prokaryotic type I topoisomerases and the ability of the enzyme to recognise single-stranded and duplex DNA are unique characteristics of the enzyme. We have mapped the strong topoisomerase sites of the enzyme on genomic DNA sequences from Mycobacterium tuberculosis and M.smegmatis. The enzyme does not nick DNA in random fashion and DNA cleavage occurred at a few specific sites. Mapping of these sites revealed conservation of a pentanucleotide motif CG/TCT↓T at the cleavage site (↓ represents the cleavage site). The enzyme binds and cleaves consensus oligonucleotides having this sequence motif. The protein exhibits a very high preference for C or a G residue at the +2 position with respect to the cleavage site. Based on earlier and the present studies we propose that the enzyme functions in vivo mainly at these specific sites to carry out topological reactions.     

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.     

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.     

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.     

Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro

A series of synthetic peptides representing authentic proteolytic cleavage sites of human rhinovirus type 14 were assayed as substrates for purified 3C protease. Competition cleavage assays were employed to determine the relative specificity constants (Kcat/Km) for substrates with sequences related to the viral 2C-3A cleavage site. Variable length peptides representing the 2C-3A cleavage site were cleaved with comparable efficiency. These studies defined a minimum substrate of 6 amino acids (TLFQ/GP), although retention of the residue at position P5 (ETLFQ/GP) resulted in a better substrate by an order of magnitude. Amino acid substitutions at position P5, P4, P1', or P2' indicated that the identity of the residue at position P5 was not critical, whereas substitutions at position P4, P1' or P2' resulted in substrates with Kcat/Km values varying over 2 orders of magnitude. In contrast to the 2C-3A cleavage site, small peptide derivatives representative of the 3A-3B cleavage site were relatively poor substrates, which suggested that residues flanking the minimum core sequence may influence susceptibility to cleavage. The 3C protease of rhinovirus type 14 was also capable of cleaving peptides representing comparable cleavage sites predicted for coxsackie B virus and poliovirus.     

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

Restricted fragmentation of poliovirus type 1, 2 and 3 RNAs by ribonuclease III.

Cleavage of the genome RNAs of poliovirus type 1, 2 and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack “primary cleavage sites”; (2) lowering the salt concentration to 0.1 m or below allows RNase III to cleave the RNAs at “secondary sites”. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)- linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.     

Cloning and characterization of a lysosomal phospholipase A2, 1-O-acylceramide synthase.

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A(2) and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A(2) activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized with N-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.     

DNA cleavage by type III restriction-modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites

The type III restriction-modification enzyme EcoP15I requires the interaction of two unmethylated, inversely oriented recognition sites 5'-CAGCAG in head to head configuration to allow an efficient DNA cleavage. It has been hypothesized that two convergent DNA-translocating enzyme-substrate complexes interact to form the active cleavage complex and that translocation is driven by ATP hydrolysis. Using a half-automated, fluorescence-based detection method, we investigated how the distance between two inversely oriented recognition sites affects DNA cleavage efficiency. We determined that EcoP15I cleaves DNA efficiently even for two adjacent head to head or tail to tail oriented target sites. Hence, DNA translocation appears not to be required for initiating DNA cleavage in these cases. Furthermore, we report here that EcoP15I is able to cleave single-site substrates. When we analyzed the interaction of EcoP15I with DNA substrates containing adjacent target sites in the presence of non-hydrolyzable ATP analogues, we found that cleavage depended on the hydrolysis of ATP. Moreover, we show that cleavage occurs at only one of the two possible cleavage positions of an interacting pair of target sequences. When EcoP15I bound to a DNA substrate containing one recognition site in the absence of ATP, we observed a 36 nucleotide DNaseI-footprint that is asymmetric on both strands. All of our footprinting experiments showed that the enzyme did not cover the region around the cleavage site. Analyzing a DNA fragment with two head to head oriented recognition sites, EcoP15I protected 27-33 nucleotides around the recognition sequence, including an additional region of 26 bp between both cleavage sites. For all DNA substrates examined, the presence of ATP caused altered footprinting patterns. We assume that the altered patterns are most likely due to a conformational change of the enzyme. Overall, our data further refine the tracking-collision model for type III restriction enzymes.     

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.     

Resolution of Holliday junction substrates by human topoisomerase I

Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.     

Recognition sequence of a restriction endonuclease from Haemophilus gallinarum

From comparison of the sequences in and around the cleavage sites of restriction endonuclease HgaI isolated from Haemophilus gallinarum, the recognition sequence and cleavage site of this enzyme was deduced as below: (formula: see text) This enzyme recognizes a specific but asymmetric penta-nucleotide sequence, GCGTC, and introduces staggered cleavages at appointed positions away from the recognition sequence, generating protruding 5'-ends of five nucleotides. The sequences surrounding the cleavage sites bear no obvious relation to one another.     

The proteomics of N-terminal methionine cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1'), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1' were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2', P3', and P4' strongly slowed the reaction, and some proteins with Val and Thr at P1' could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought.