Programmable cleavage of linear double-stranded DNA by combined action of Argonaute CbAgo from Clostridium butyricum and nuclease deficient RecBC helicase from E. coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecB^exo–C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecB^exo–C were thoroughly explored using 0.03–25 kb dsDNAs. When combined with RecB^exo–C, CbAgo could cleave targets located 11–12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecB^exo–C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecB^exo–C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.     

Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis

Argonaute (Ago) proteins are conserved nucleic acid-guided proteins present in all domains of life. Eukaryotic Argonaute proteins (eAgos) are key players in RNA interference pathways and function as RNA-guided RNA endonucleases at physiological temperatures. Although eAgos are considered to evolve from prokaryotic Argonaute proteins (pAgos), previously studied pAgos were unable to catalyze RNA-guided RNA cleavage at physiological temperatures. Here, we describe a distinctive pAgo from mesophilic bacteria Kurthia massiliensis (KmAgo). KmAgo utilizes DNA guides to cleave single-stranded DNA (ssDNA) and RNA targets with high activity. KmAgo also utilizes RNA guides to cleave ssDNA and RNA targets at moderate temperatures. We show that KmAgo can use 5′ phosphorylated DNA guides as small as 9-mers to cut ssDNA and RNA, like Clostridium butyricum Ago. Small DNA binding confers remarkable thermostability on KmAgo, and we can suppress the guide-independent plasmid processing activity of empty KmAgo by elevating the DNA guide loaded temperature. Moreover, KmAgo performs programmable cleavage of double-stranded DNA and highly structured RNA at 37°C. Therefore, KmAgo can be regarded as a DNA-guided programmable omnipotent nuclease for cleaving most types of nucleic acids efficiently. This study broadens our understanding of Ago proteins and could expand the pAgo-based DNA and RNA manipulation toolbox.     

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100°C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80–160 mM NaCl and 70°C, PaNth displays DNA glycosylase/β-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.     

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.     

Cloning,sequence analysis and expression in E. coli of the DNA polymerase I gene from Chloroflexus aurantiacus,a green: nonsulfur eubacterium

We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli. One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E. coli. Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3′-5′ exonuclease domain is 30% identical to the corresponding E. coli domain and that three sequence motifs associated with 3′-5′ exonuclease activity are conserved. Also, a protein fraction from E. coli expressing the Chloroflexus polymerase contains a thermostable 3′-5′ exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis. The N-terminal 5′-3′ exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E. coli domains and all sequence motifs associated with these two enzymatic activities also are conserved. Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3′-5′ exonuclease domain was introduced later in those eubacterial branches that have this activity. Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution.     

CUT Domains Stimulate Pol β Enzymatic Activities to Accelerate Completion of Base Excision Repair

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5′-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.     

The bacteriophage P1 restriction endonuclease

The bacteriophage P1 restriction endonuclease has been purified from Escherichia coli lysogenic for P1. This restriction endonuclease P has a sedimentation coefficient of 9.3 S. Unlike the E. coli K restriction endonuclease, endonuclease P does not require S-adenosylmethionine for breakage of DNA. S-adenosylmethionine does, however, stimulate the rate of double-strand breakage of DNA by endonuclease P. Hydrolysis of ATP by endonuclease P could not be detected under conditions in which the K restriction endonuclease massively degrades ATP.The enzyme makes a limited number of double-strand breaks in unmodified or heterologously modified λ DNA. In the presence of S-adenosylmethionine, it does not cut every DNA molecule to the same extent. Incubation of λ DNA with excess amounts of enzyme in the presence of S-adenosylmethionine results in less breakage of the DNA than with smaller amounts of enzyme. This effect is not seen in the absence of S-adenosylmethionine. The maximum amount of cutting in the absence of S-adenosylmethionine appears to be greater than the maximum amount of cutting in its presence. This is most likely due to the modification methylase activity of P1 restriction endonuclease.     

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E.coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3′- or 5′-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5′-terminus of the 3′ cleaved fragment, but is unable to remove DHU remaining on the 3′-terminus of the cleaved 5′ fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3′-terminus of a cleaved 5′ fragment, but are unable to remove DHU remaining on the 5′-terminus of a cleaved 3′ fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Massively parallel characterization of restriction endonucleases

Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site–flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.     

Purification of a hybrid molecule composed of tyrosine transfer RNA and its complementary DNA sequence

The transducing bacteriophage φ80psu_III⁺ carries one structural Escherichia coli gene specifying tyrosine tRNA.The r strand of bacteriophage φ80psu_III⁺ was hybridized with E. coli transfer RNA and the hybrid digested with Neurospora crassa endonuclease. The analysis of the products of enzymic digestion demonstrated the release of a cistron-hybrid composed of tyrosine tRNA and its complementary DNA sequence. The cistron-hybrid was purified from unhybridized DNA by cesium sulphate density-gradient centrifugation and gel filtration.The ratio between tyrosine tRNA and its complementary DNA sequence in the final product was 1:1 as demonstrated by radioisotopic analysis. This purification represents a 30,000-fold enrichment of the E. coli genome for a specific DNA sequence.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16–20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.     

Investigating the endonuclease activity of four Pyrococcus abyssi inteins

Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases. Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity. PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site. This is consistent with their size and with the relative positions and sequences of their endonuclease motifs. However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins. In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously. Furthermore, the two inteins interact differently with DNA. In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1. Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity. Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.