Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

Role of genes 46 and 47 in bacteriophage T4 reproduction. II. Formation of gaps on parental DNA of polynucleotide ligase defective mutants

The nature of nucleolytic activity regulated by genes 46 and 47 of bacteriophage T4 was studied by examining the metabolism of parental DNA of phages carrying a mutation in polynucleotide ligase gene (lig) and an additional mutation in one of the following D0 genes (D0 genes are necessary for T4 DNA synthesis): 32, 43 (DNA polymerase  pol), 44 and 45. Polynucleotide ligase and DNA polymerase were used to distinguish nicks (phosphodiester bond interruptions on duplex DNA) from gaps (interruptions with missing nucleotides). In non-permissive hosts, parental DNA of double mutants (lig, D0) accumulated both single- and double-strand breaks. Up to 30% of this DNA eventually became acid-soluble. An additional mutation in gene 46 (or 47) did not prevent accumulation of double- and single-strand breaks but did prevent degradation to the acid-soluble state. The majority of the single-strand breaks on (lig, D0)-DNA were presumed to be gaps since, after extraction from infected host cells, they were repaired by ligase plus DNA polymerase but not by ligase alone. In contrast, the majority of the single-strand breaks on parental DNA of (lig, D0, 46) or (lig, pol, 47) were repaired by ligase alone, suggesting nicks, rather than gaps. These observations suggest that (i) genes 46 and 47 regulate, either directly or indirectly, an exonuelease activity which can attack T4 DNA at nicks to create gaps, and (ii) T4 DNA polymerase, and the products of genes 32, 44 and 45 are necessary to prevent nicks from becoming gaps in vivo. Possible roles for genes 46 and 47 in T4 DNA replication and in recombination are discussed.     

Effect of lig-7 on strand joining in repair of damaged DNA and on cutting of intact homologous DNA (cutting in trans) in Escherichia coli

Genetic recombination in Escherichia coli depends on the recA⁺ gene and can be increased in frequency by certain treatments that damage DNA. In previous studies (Ross &; Howard-Flanders, 1977a,b), E. coli (λ) cells were infected with undamaged λ phages and then with λ phages that were either undamaged, or had interstrand crosslinks produced in their DNA by treatment with psoralen and light. When the superinfecting DNA contained psoralen crosslinks, the intact DNA was cut. This cutting, referred to as cutting in trans, occurred only in DNA genetically homologous to the damaged DNA, required recA⁺ and behaved as expected of a step in damage-induced genetic recombination.In the present studies, we investigated the effect on cutting in trans of lig-7, a thermosensitive allele of the structural gene for E. coli polynucleotide ligase and also of uvrA, which controls the excision of damaged bases from DNA. The ligase deficiency caused gaps due to the action of the uvrA⁺ endonuclease on damaged DNA to remain open for at least 25 minutes. For low levels of damage, cutting in trans was also enhanced in the lig-7 cells at non-permissive temperatures but was not increased in wild-type cells. The enhanced cutting in trans depended upon genetic homology, as expected if it reflected elevated levels of damage-induced genetic recombination. Presumably, the unrepaired gaps in the damaged DNA made it a good substrate for the enzymes that promote cutting in trans of its homologs.     

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.     

Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli

Summary The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5-phosphoryl and 3-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690.On leave from Takara Shuzo Co., Ltd., Kyoto, Japan     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1

DNA ligases join 3′ hydroxyl and 5′ phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.     

Number and size distribution of the DNA chains in Escherichia coli.

The essential replication protein encoded by gene O of bacteriophage λ (O-λ) is one of the major polypeptides produced in vitro in a DNA-dependent protein synthesizing system with λ DNA as template (Yates et al., 1977). We have used this system to identify the proteins encoded by lambdoid phages φ80 and 82 and equivalent in function to O-λ. The O protein of each phage type differs slightly in polypeptide molecular weight. Hybrid λ-φ80 and λ-82 phages derived by recombination within gene O direct synthesis of hybrid O proteins with the aminoterminal segment characteristic of one parent, and the carboxyl-terminal segment characteristic of the other. Differences in structure among O-λ, O-80 and O-λ82 segregate together with specificity determinants for interactions between the O protein and the control site ori, and between the O protein and the product of replication gene P. The coding region for the O protein includes ori.     

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.     

Effect of chloramphenicol and the recB gene product on DNA metabolism in Escherichia coli K12 strains defective in DNA ligase

Summary We have examined DNA strand breakage, DNA degradation, and the rate of DNA synthesis in lig and lig-recB strains of Escherichia coli K12 incubated in the presence and absence of 3 g/ml chloramphenicol. Substantial DNA strand breakage and DNA degradation is observed in the lig strain upon growth at 40°C; however, such strand breakage and DNA degradation is not observed in the lig-recB strain. Incubation of the lig strain at 40°C in the presence of 3 g/ml chloramphenicol reduces the amount of DNA strand breakage and DNA degradation to the level observed in the lig-recB strain. Together, these results demonstrate that exonuclease V (the recBC gene product) is responsible for the increased DNA degradation associated with DNA ligase deficiency.     

DNA degradation in an amber mutant of Escherichia coli K12 affecting DNA ligase and viability

Isolation of an amber mutant lig-321 (or dnaL321) if Escherichia coli K12 with a defect in DNA ligase activity was previously reported (Nagata & Horiuchi, 1974). This was the first demonstration that, in E. coli, conditionally lethal nonsense mutants can be isolated selectively. Unlike the hitherto available E. coli K12 DNA ligase-deficient (lig) mutants, the DNA of this mutant is degraded under lethal conditions. This paper describes its further characterization. The DNA degradation was found to be an energy-requiring process, in which endonuclease I did not seem to participate. Kinetic analyses of prelabeled DNA indicated that the parental strands were degraded. The sedimentation profile of prelabeled DNA in an alkaline sucrose gradient showed that the extensive degradation was preceded by a step in which the parental strands were broken into relatively large pieces. At least in the early phase of degradation, which we examined by alkaline sucrose gradient centrifugation of pulse-labeled DNA, synthesis of discontinuous daughter chains (Okazaki fragments, Okazaki et al., 1968) was confirmed. Joining of the nascent chains, however, was completely inhibited. Genetic analyses revealed that the mutant allele is recessive to the wild type. This agrees with in vitro studies in which the mutant crude extract was found not to inhibit DNA ligase activity of the wild type extract. These and other properties of the lig-321 mutant were compared with the other DNA ligase-deficient mutants of E. coli. The role of this enzyme in DNA replication, repair and recombination is discussed.     

A kinetic analysis of DNA ejection from tailed phages revealing the prerequisite activation energy

All tailed bacteriophages follow the same general scheme of infection: they bind to their specific host receptor and then transfer their genome into the bacterium. DNA translocation is thought to be initiated by the strong pressure due to DNA packing inside the capsid. However, the exact mechanism by which each phage controls its DNA ejection remains unknown. Using light scattering, we analyzed the kinetics of in vitro DNA release from phages SPP1 and λ (Siphoviridae family) and found a simple exponential decay. The ejection characteristic time was studied as a function of the temperature and found to follow an Arrhenius law, allowing us to determine the activation energy that governs DNA ejection. A value of 25-30 kcal/mol is obtained for SPP1 and λ, comparable to the one measured in vitro for T5 (Siphoviridae) and in vivo for T7 (Podoviridae). This suggests similar mechanisms of DNA ejection control. In all tailed phages, the opening of the connector-tail channel is needed for DNA release and could constitute the limiting step. The common value of the activation energy likely reflects the existence for all phages of an optimum value, ensuring a compromise between efficient DNA delivery and high stability of the virus.     

Cloning and expression of a new bacteriophage (SHPh) DNA ligase isolated from sewage

During the last 50 years, major advances in molecular biology and biotechnology have been attributed to the discovery of enzymes that allow molecular cloning of important genes. One of these enzymes that has been widely acknowledged for its role in the development of biotechnology is the T4 DNA ligase. This enzyme joins the break in the DNA backbone structure by creating a phosphodiester bond between 5′ PO₄ and 3′ OH ends, in an ATP dependent multi-step reaction, thus allowing the ligation of related and foreign DNA sequences. Due to its role in modern DNA recombinant technology, there is a high demand on DNA ligase to allow the ligation of target DNA inserts into a chosen vector as part of DNA cloning technology. To closely look at ligase sequence diversity, a bacteriophage that infects DH5α (commercial lab strain of Escherichia coli) was isolated from sewage system in Hebron, Palestine. The DNA ligase gene of this phage was cloned and its sequence was compared to the NCBI database. The new bacteriophage ligase, named (South Hebron Phage, SHPh) DNA ligase, shows homology to T even bacteriophage DNA ligases posted in the NCBI database with 35 nucleotide differences, an indication of existed diversity among T even DNA ligation enzymes that can be used as markers in phage classification.     

Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.     

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT?-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris–HCl. The activity was highly dependent on MgCl₂ or MnCl₂ with maximal activity above 5 mM MgCl₂ and 2 mM MnCl₂. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.