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PubMed1976

Biochemical construction of specific chimeric plasmids from ColE1 DNA and unfractionated Escherichia coli DNA.

A series of chimeric plasmids was constructed using colicinigenic factor E1 (ColE1) DNA as the replicon and DNA fragments carrying the galactose or tryptophan operons from E. coli. Restriction endonuclease EcoRI digests of ColE1 DNA and various DNAs containing the trp or gal operons were joined by T4 polynucleotide ligase [polynucleotide synthetase (ATP), poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1]. Chimeric plasmids carrying the desired genes were selected after transformation of Trp- or Gal- cells with ligated DNA. By using this method, we constructed ColE1-gal and ColE1-trp chimeric plasmids in which the source of the bacterial gal and trp operons was an unfractionated EcoRI digest of total E. coli DNA. The frequency of recovery of such chimeric plasmids is 10 to 20 colonies per mug of ligated DNA used in the transformation step. The method utilized in this report for constructing specific chimeric plasmids from total E. coli DNA is very simple. It requires only endonuclease R-EcoRI and T4 polynucleotide ligase, both of which are commercially available. The yield of transformants suggests that this method will be useful for cloning and amplifying a wide variety of functionally defined genes from E. coli and other prokaryotic organisms.

Colicins↗
PubMed1970

Polynucleotide ligase-catalyzed joining of deoxyribo-oligonucleotides on ribopolynucleotide templates and of ribo-oligonucleotides on deoxyribopolynucleotide templates.

T4 polynucleotide ligase efficiently catalyzes the head-to-tail joining of the ribo-oligoadenylates, r-(pA)(8) and r-(pA)(10), in the presence of high molecular weight deoxypolythymidylate. The enzyme also catalyzes the joining of deoxy-oligothymidylates, e.g., d-(pT)(10), in the presence of ribopolyadenylate. The enzyme failed to bring about the joining of r-(pA)(10) when poly r-U was used as the template, although a slow formation of the expected activated intermediate from r-(pA)(10) was detected.

Adenine Nucleotides↗
PubMed1990

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison.

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1976

In vitro repair of radiation-induced strand breaks in DNA.

DNA, gamma-irradiated in vitro or in isolated thymocytes was treated with several enzymes to achieve repair of the radiation-induced single strand braks. Whereas an incubation with polynucleotide ligase can join only 25% of the single strand breaks, a combined treatemnt with exonuclease III (EC 3.1.4.1), DNA polymerase I (EC 2.7.7.7), and polynucleotide ligase leads to repair of 80% of the breaks. For this in vitro repair the exonuclease III has to remove several, probably damaged, nucleotides from the 3'-terminal producing a single-stranded gap, which will be filled in by DNA polymerase I and joined by ligase. Tests for successful rejoining of the strand breaks were performed by showing the loss of primer 3'-OH sites for DNA polymerase I, by the resistance of incorporated nucleotides in the gap to removal by a second exonuclease III treatment, and by strand break determination in the analytical ultracentrifuge. 20% of the radiation-induced strand breaks will not be repaired by this combined treatment possibly due to an incomplete binding of the ligase on the 5'-terminals and/or an incomplete removal of the damaged 3'-terminals by exonuclease III.

Animals↗
PubMed2003

Structure-function analysis of T4 RNA ligase 2.

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.

Adenosine Triphosphate↗
PubMed1975

In vitro repair of UV-or x-irradiated bacteriophage T4 DNA by extract from blue-green alga Anacystis nidulans.

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.

Adenosine Triphosphate↗
PubMed1972

Enzymatic oligomerization of bacteriophage P22 DNA and of linear Simian virus 40 DNA.

Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers, but higher oligomers are also present.DNA ligase extracted from uninfected Escherichia coli seems unable to perform a similar reaction, which is concluded to involve the fully base-paired termini. Linear double-stranded molecules of simian virus(SV) 40 DNA, produced by the action of the bacterial restriction endonuclease R(1), are oligomerized by either ligase; therefore, this reaction seems to involve single-stranded cohesive ends. No mixed products could be found when P22 DNA and linear SV 40 DNA were exposed together to the T4 ligase.

Carbon Isotopes↗
PubMed1984

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.

Coliphages↗
PubMed1978

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.

Coliphages↗
PubMed1985

The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage.

The CDC9 gene of Saccharomyces cerevisiae encodes a DNA ligase, and we have determined the nucleotide sequence of a 3.85 kb fragment of DNA which encompasses the convergently transcribed CDC9 and CDC36 genes. S1 nuclease mapping has revealed a major 5' end for the CDC9 mRNA, and one major and one minor site for 3' polyadenylation. These two sites lie within the C-terminal coding region of the CDC36 gene, implying that these two genes are transcribed from overlapping sequences. An interesting structural feature of the CDC9 gene is a series of 6 hexanucleotide repeats (ATGATT) which occur within the 650 bp immediately upstream from the site of transcription initiation. These repeat elements may be implicated in the cell division cycle regulated expression of CDC9. Comparison of the predicted amino acid sequence of the yeast DNA ligase (Mr 84,806) with the sequences of the T4 and T7 bacteriophage DNA ligases reveals little similarity except for a stretch of approximately 45 amino acids, comprising 3 short homologous segments. This region may represent an ATP-binding domain common to polynucleotide ligases.

Amino Acid Sequence↗
PubMed1975

Excision-repair of gamma-ray-damaged thymine in bacterial and and mammalian systems.

The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t') from gamma-irradiated or OSO4-oxidized DNA or synthetic poly[d(A-T)] was observed with crude extracts of Escherichia coli and isolated nuclei from human carcinoma HeLa S-3 and Chinese hamster ovary cells. The results with E. coli extracts allow the following conclusion: (1) The uvrA-gene product is not required for t' excision. (2) Radiation-induced strand breakage is not required for product excision. (3) Experiments with extracts of E. coli polAexl showed that the 5' in equilibrium 3' exonuclease associated with polymerase I is responsible for the removal of t'. (4) Experiments with extracts of E. coli endo I lig 4 and the ligase inhibitor nicotinamide mononucleotide showed that polynucleotide ligase accomplishes the last strand resealing step in the excision-repair of t'. Isolated nuclei from HeLa and Chinese hamster ovary cells possess the necessary enzymes for the selective excision of t' from gamma-irradiated or osmium tetroxide oxidized DNA. Approximately 25 to 35% of the products were removed from DNA within 60 min. Unspecific DNA degradation was very low. Radiation-induced strand breakage is not required for product removal.

Cell Line↗
PubMed1977

Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme.

Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

Coliphages↗