A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.     

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4-infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk- or rli- infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4-induced anticodon nuclease cleaves lysine tRNA 5' to the wobble position, yielding 2':3'-P greater than and 5'-OH termini. Polynucleotide kinase converts them into a 3'-OH and 5' P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.     

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO₂-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5′-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa > Gua > adenine (Ade) ∼ thymine (Thy) > cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Purification and electrophoretic assay of T4-induced polynucleotide ligase for the in vitro construction of recombinant DNA molecules

A facile method for the determination of bacteriophage T4-induced polynucleotide ligase joining activity is described. The assay is based on the ability of polynucleotide ligase to join the cohesive termini of bacteriophage λ DNA covalently. The observance of this activity is greatly facilitated if λ DNA is previously cleaved with the restriction endonuclease EcoRI and the reaction products subsequently analyzed by electrophoresis in ethidium bromide-agarose gel. A purification scheme is presented which offers a reduction in the number of steps required to purify polynucleotide ligase compared to a previously published procedure and yields an enzyme preparation which is suitable for use in in vitro construction of recombinant DNA molecules.     

A rapid and accurate procedure for assaying DNA polymerase,RNA polymerase,or ribosome dependent protein synthesis

A descending paper chromatographic procedure for separating trichloracetic acid soluble from precipitable material is described. Its use for assaying DNA, RNA, or protein synthesis from a variety of sources with various states of purity attests to its general applicability to reactions of interest to molecular biologists. Large numbers of analyses can be carried out in a short period of time without compromising accuracy or reliability. The use of this procedure for still more enzymes, those involved in modifying DNA, RNA, or proteins, is discussed.     

Purification and properties of bacteriophage T4-induced RNA ligase

RNA ligase has been extensively purified by a new procedure in high yield from T4-infected Escherichia coli. The enzyme consists of a single polypeptide chain of molecular weight 47,000. It catalyzes the formation of a phosphodiester bond between a 5′-PO₄-terminated oligonucleotide and a 3′-OH terminated oligonucleotide. The purified enzyme catalyzes both the intramolecular formation of single-stranded circles with longer oligonucleotides of the type pAp(Ap)_nA_?OH, where n is about 15 or greater and the intermolecular joining of pAp(Ap)₃A_OH (where the 5′-PO₄-terminated oligonucleotide is short enough to prevent apposition of its 3′ and 5′ ends) to UpUpU_OH when high concentrations of the 3′-OH-terminated acceptor oligonucleotide are present. Preparations of RNA ligase at all stages of purification show an unusual dependence of specific activity of the enzyme on the concentration of enzyme present in the assay. However, when care is taken to determine meaningful specific activities at each step, the ligase is found to be very stable during chromatography on various ion-exchange columns and may be purified by conventional techniques.     

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.     

Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.