Polynucleotide Ligase Research Articles

A polynucleotide ligase from polyoma virus-infected mouse cells.

Research Article| December 01 1968 A polynucleotide ligase from polyoma virus-infected mouse cells P Beard; P Beard 1Institutes of Virology and of Biochemistry, University of Glasgow Search for other works by this author on: This Site PubMed Google Scholar J D Pitts J D Pitts 1Institutes of Virology and of Biochemistry, University of Glasgow Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1968) 110 (4): 48P. https://doi.org/10.1042/bj1100048Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation P Beard, J D Pitts; A polynucleotide ligase from polyoma virus-infected mouse cells. Biochem J 1 December 1968; 110 (4): 48P. doi: https://doi.org/10.1042/bj1100048Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1968 The Biochemical Society1968 Article PDF first page preview Close Modal You do not currently have access to this content.

DNA replication in vivo by a temperature-sensitive polynucleotide ligase mutant of T4.

DNA replication in vivo as function of temperature sensitive polynucleotide ligase mutant Th, tsA80 in strand synthesis

Polynucleotide ligase from myeloid and lymphoid tissues.

Polynucleotide ligase from myeloid and lymphoid tissues.

Enzymatic Breakage and Joining of Deoxyribonucleic Acid: VII. PROPERTIES OF THE ENZYME-ADENYLATE INTERMEDIATE IN THE POLYNUCLEOTIDE LIGASE REACTION

Polynucleotide ligase from Escherichia coli infected with bacteriophage T4 catalyzes the esterification of interrupted phosphodiester bonds (single strand breaks) within a DNA duplex. Purified preparations of the enzyme also catalyze an isotopic exchange reaction between ATP and 32PPi. However, they do not catalyze a measurable exchange between 3H-AMP and ATP. A radioactively labeled ligaseAMP complex has been isolated by gel filtration and its properties have been studied. Although purified preparations of polynucleotide ligase require ATP for the esterification of interrupted phosphodiester bonds in a DNA substrate, preparations of ligase-AMP can form phosphodiester bonds in the absence of added ATP. Release of AMP occurs during this reaction and is dependent on the presence of 5′-phosphomonoesters at single strand breaks in the DNA duplex. The extent of the release of AMP is proportional to the number of internal 5′-phosphomonoesters present in the reaction mixture. Native T7 DNA, denatured DNA, sonically treated DNA, and DNA containing only 5′-hydroxyl end groups produce little, if any, ligase-AMP breakdown. The DNA-dependent breakdown of the ligase-AMP complex provides a sensitive method for determining the number of internal 5′-phosphomonoesters in preparations of duplex DNAs.

Enzymatic Breakage and Joining of Deoxyribonucleic Acid: VI. FURTHER PURIFICATION AND PROPERTIES OF POLYNUCLEOTIDE LIGASE FROM ESCHERICHIA COLI INFECTED WITH BACTERIOPHAGE T4

Abstract Polynucleotide ligase has been purified 1000-fold from extracts of Escherichia coli infected with bacteriophage T4. The enzyme catalyzes (a) the repair of phosphodiester bond scissions introduced into the strands of bihelical DNA by pancreatic DNase and (b) the conversion of hydrogen-bonded circular duplexes or concatemers of λ bacteriophage DNA to covalently bonded circular molecules or concatemers, respectively. The DNA products of these reactions have been characterized by sedimentation and end group analysis. The ligase does not catalyze the end to end joining of T7 bacteriophage DNA. The standard assay for ligase activity measures the protection of a 32P-labeled 5'-phosphoryl group, located at a single strand break in DNA, from attack by bacterial alkaline phosphatase. The enzyme purification procedure has been redesigned to eliminate the necessity for enzyme assays during the early fractionation steps and to permit the use of a simple assay, based on ATP-PPi exchange, during the later stages of purification. The ligase reaction has a specific requirement for a bihelical DNA substrate and for ATP, and it results in the formation of phosphodiester bonds, AMP, and pyrophosphate. The Km for DNA containing single strand breaks is 1.5 x 10-9 m expressed as the concentration of internal phosphomonoesters. The Km for ATP is 1.4 x 10-5 m, and dATP is a competitive inhibitor of the enzyme (Ki of 3.5 x 10-5 m).

Mechanism of DNA chain growth, II. Accumulation of newly synthesized short chains in E. coli infected with ligase-defective T4 phages.

The most recenttly replicated portion of the bacterial anid T4 phage chromosome, selectively labeled by ani extremely short radioactive pulse, can be isolated, after denaturation, as short DNA chains with ain average sedimentation coefficient of 7--11 _S-3 ' This supports an hypothesis of discontinuous DNA chain growth, by whicli short stlretches of DNA are synlthesized at the replicatinig point aid subsequently connected to the older portion of the growinig strands by formationl of phosphodiester linkages.21The joiling of these short chainis is assumed to be carried out by polyntucleotide ligase, a-n en1zyme discovered recently in ni-orlmal anld T4 phage-infected -. c1oi.712 If DNA replicates in vivo by such a nmechanism, the n1e-wly synthesized shorit DNA chainis would accumulate in the cell uitder conditions where the function of ligase is temporarily impaired. In the present work, this predictiolt was tested aIld velified by experimenIts with T4 p-iwage mutants, which produce therinosensitive polynueleotide ligase.3 ifaterials and ethlods.----Bacteria and bacteriophages: Escherichia coli 11/5, bacteriophage T4 ts A80, and T4 ts B20 were generous gifts of Dr. R. S. Edgar. T4 ts A80 and T4 ts 1820 are gene 30 mutants, which induce temperature-sen tsitive polynlucleotide ligase.13 T4D) (wild-typ.e) was supplied by Dr. J. Tomlizawa. Infection of bacteriophages: E. coli 1B/5 was grown in a glucose salt medium (MAedium A described previously3) at 370 to 5 X -108 cells/ ml, and incubated at 20? for 20 mill. DI-tryptophan (40 Aug/ml) anld bacteriolhages (m.o.i. -1(0) were thenl added to the culture and the incubation was continued at 200 with shaking. Temperature shift: When the volume of a culture was 10 ml or less, temaperature shift from 200 to 30-44? was attained simply by pouring the culture into a flask bein-Ig shaken in a water bath of the new temperature. For larger volumes, the culture was warmed to a new teruperature by beirng shaken in a 50? water bath before being placed in the water bath of the new temperature. 'By these methods temperature shift-up was complete(d in I mim. For temperature shift-down, the culture was chilled in ice water to the desired temiperature. It took about 10 sec to change the temnperatuire of a 30-ml culture from 43 to 300. Other methods: Pulse-labeling with H3-thym-idine, DNA extr-action, anld alkaline sucrose gradient se(limentation were carried out as described previously.3 Results.-Pulse-labeling at 43-440: Our previous stlldy (ref. 3, Fig. 6) showed ilbat H1-thymidine that had been incorporated inlto T4 wild-type infected cells (lutriiLlg the period of active phage DNA synthesis (70 mmiiafter infection at 20') first appeared in short DNA chains having an average sedimentation rate of 8-9S (in alkali) before transitionl to large chains with sedimentation coefficients of 30--60S. In order to see if temrlpor al inhibition of polynucleotide ligase causes accumulationI of tile nascent short DNA chains, E. coli B/5 infected with T4 ts A80 or ts B20 (gene 30) was pulse-labeled with H3-thymidine 65--70 minutes

Repair of single strand breaks in transforming DNA by polynucleotide ligase

Repair of single strand breaks in transforming DNA by polynucleotide ligase

On the mechanism of the polynucleotide joining reaction.

Polynucleotide-joining enzymes (ligases) have been identified in uninfected and phage-infected Escherichia coli (Gellert, 1967; Olivera and Lehman, 1967a; Zimmerman et al., 1967; Gefter et al., 1967; Weiss and Richardson, 1967a; Cozzarelli et al., 1967). Both types of enzyme catalyze the synthesis of a phosphodiester bond between the 3′ hydroxyl and 5′ phosphoryl termini of DNA chains which have been properly aligned in a double-helical structure. Such enzymes are of interest because of their possible involvement in genetic recombination, ‘dark’ repair of ultraviolet-induced lesions, and in DNA synthesis. It has already been demonstrated that the polynucleotide ligase induced by phage T4 is essential for normal T4 DNA replication (Fareed and Richardson, 1967).

Repair and recombination in phage T4. II. Genes affecting UV sensitivity.

The effect of temperature sensitive (ts) mutations in five early function genes on the sensitivity of phage T4D to ultraviolet (UV) light was determined. Temperature sensitive mutations in genes 30, 43, and 47 were found to increase the sensitivity of T4D to ultraviolet light. Mutations in genes 45 and 56 did not alter UV sensitivity. Gene 30 has been identified as the structural gene for polynucleotide ligase (Fareed and Richardson, 1967); gene 43 codes for DNA polymerase (deWaard et al., 1965; Warner and Barnes, 1966); mutants in gene 47 are unable to degrade the host chromosome (Wiberg, 1966) and are probably defective in a deoxyribonuclease. Deoxycytidine triphosphatase is the enzyme specified by gene 56 (Wiberg, 1967); and the product of gene 45 has not been identified.

Intermediates in T4 DNA replication in a T4 ligase deficient strain.

We have studied the role of polynucleotide ligase in DNA replication in T4 infected cells. We have confirmed Okazaki's finding on the physical state of newly replicated DNA in phage T4 infected bacteria (Okazaki et al., 1968) and have shown that the small segments are probably precursors for high molecular weight DNA. We have also obtained evidence that the T4 induced ligase functions in the conversion of the small pieces to high molecular weight DNA.

Repair and recombination in phage T4. I. Genes affecting recombination.

In a previous paper (Bernstein, 1967) temperature-sensitive (ts) mutations in gene 43 (DNA polymerase) and gene 42 (deoxycytidylate hydroxymethylase) of phage T4 were shown to increase recombination severalfold. Evidence is presented here that a ts mutation in gene 30 also increases recombination. Gene 30 has been identified as the structural gene of polynucleotide ligase (Fareed and Richardson, 1967).

Studies on the joining of DNA by polynucleotide ligase of phage T4.

Studies on the joining of DNA by polynucleotide ligase of phage T4.

Enzymatic Breakage and Joining of Deoxyribonucleic Acid: III. AN ENZYME-ADENYLATE INTERMEDIATE IN THE POLYNUCLEOTIDE LIGASE REACTION

Abstract Polynucleotide ligase, isolated from Escherichia coli infected with bacteriophage T4, catalyzes (a) the repair of an interrupted strand in a DNA duplex by the formation of phosphodiester bonds, with a concomitant cleavage of ATP to AMP and PPi, and (b) an ATP-PPi exchange reaction which is not dependent on the presence of DNA. The two activities elute together from diethylaminoethyl cellulose and phosphocellulose. An enzyme-adenylate complex has been isolated and is found to mediate both the formation of phosphodiester bonds in the absence of ATP and the formation of ATP from PPi. These findings suggest that as the first step of the ligase reaction, ATP and enzyme react to form an enzyme-adenylate intermediate accompanied by the release of PPi.

Polynucleotide cellulose as a substrate for a polynucleotide ligase induced by phage T4

An enzyme which joins polydeoxynucleotide strands has been identified in and purified from extracts Escherichia coli infected with phage T4 am N82 and appears to be the same as the ligase described by Weiss and Richardson. A novel method for detecting and assaying such enzymes involves the joining of a strand to one that is covalently linked to cellulose.

A biological assay for polynucleotide ligase: Recovery of marker activity in DNA-transformation

A biological assay for polynucleotide ligase: Recovery of marker activity in DNA-transformation

Enzymatic breakage and joining of deoxyribonucleic acid. II. The structural gene for polynucleotide ligase in bacteriophage T4.

Enzymatic breakage and joining of deoxyribonucleic acid. II. The structural gene for polynucleotide ligase in bacteriophage T4.

Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage.

Enzymatic breakage and joining of preformed polynucleotide strands has been implicated in molecular recombination, the repair of ultraviolet-irradiated DNA, and the interconversion of linear and circular DNA molecules.' A possible intermediate, in each of these processes, is a duplex DNA molecule containing phosphodiester bond interruptions (single-strand breaks). This paper describes the purification and some properties of an enzyme system, polynucleotide ligase, which catalyzes the repair of single-strand breaks by the formation of phosphodiester bonds (Fig. 1). The enzyme system has been purified from Escherichia coli infected with T4 bacteriophage and found to require ATP for activity.