Streptovaricins inhibit Focus Formation by MSV (MLV) Complex

We recently reported that the streptovaricins inhibit the reaction by which DNA is transcribed from the RNA template resident in murine leukaemia virions (MLV)¹. The reports^{2, 3} which first indicated that this DNA polymerase is present in oncogenic RNA viruses have been confirmed and extended^4–8. The enzyme provides a mechanism whereby an RNA virus can insert stable genetic information into a host chromosome. Gallo and co-workers described an RNA dependent DNA polymerase in lymphoblastic leukaemic cells which was inhibited by N-demethylrifampicin⁹ and this antibiotic, together with a number of other rifamycin derivatives, also inhibited the oncogenic viral DNA polymerase¹⁰. Like the streptovaricins, the rifamycins are ansa macrolide antibiotics (Fig. 1).     

The presence of terminal deoxynucleotidyl transferase in the N-methyl-N-nitrosourea induced leukaemia in BDF1 mice and its effect on the accuracy of the DNA polymerases.

DNA polymerases have been prepared from leukaemic and normal spleens and their fidelity in copying a polyd AT).polyd(AT) template assessed. The leukaemic cytoplasmic DNA polymerases were less accurate than the controls whereas no difference in accuracy was observed in the nuclear DNA polymerases. The preparations of leukaemic cytoplasmic DNA polymerase also contained the enzyme terminal deoxynucleotidyl transferase. When this enzyme was removed by further purification the accuracy of the cytoplasmic DNA polymerases increased to that of the controls.     

Fidelity of deoxyribonucleic acid polymerases from normal and leukaemic human cells in polydeoxynucleotide replication (Short Communication)

Although the relative incorporation of incorrect nucleotide (dCTP or dGTP) into poly(dA-dT).poly(dA-dT) by partially purified 3-4S DNA polymerase from normal or leukaemic human cells was four to five times higher than that by the 6-7S DNA polymerase, no significant differences in the infidelity of these polymerases between normal and leukaemic cells were noted.     

Competition for the DNA template between RNA polymerase molecules from normal and phage-infected E. coli

Summary Preincubation of E. coli core RNA polymerase lacking sigma-factor with limiting amounts of T2-DNA markedly decreases subsequent synthesis of RNA by RNA polymerase holoenzyme. Hence, although the core binds to DNA more weakly than does the holoenzyme, it can actively compete with RNA polymerase for the DNA template.Both core RNA polymerase and holoenzyme from uninfected bacteria are effective in competition with RNA polymerase isolated from T2-infected cells. On the other hand the enzyme obtained from T2-infected cells compete weakly with RNA polymerase from E. coli. The incubation of bacterial core-enzyme with a supernatant protein fraction obtained from phage-infected bacteria lowers its ability to compete with normal RNA polymerase for DNA template.These results are discussed from the viewpoint that in certain cases the RNA polymerase itself can act as a kind of repressor, effecting negative regulation of RNA synthesis. The modification of core and formation of anti-sigma induced by bacteriophage could participate in such kind of regulation.     

RNA synthesis in nuclei of rat liver and of human lymphocytes.

Physico-chemical properties and RNA synthesis in the rat liver and human lymphocytes have been compared in a nuclear system in vitro. Human lymphocytes were isolated from blood of healthy donors and of chronic lymphocytic leukaemia patients. The isolated nuclei served as the source of polymerase and template DNA. 3H-CTP was incorporated into the acid insoluble fraction linearly for 60 min. The nuclei of lymphocytes contained small amounts of RNA and protein, and the isolation procedure was complicated. Rat liver nuclei seem to be less prone to clumping at high pH values and may incorporate much more 3H-CTP. The nuclear synthesis was compared with incorporation of 3H-rU and 32P-orthophosphate into nuclear RNA of intact lymphocytes. Normal cells easily incorporated 32-P, and in contrast leukaemic cells incorporated 3H-rU to a greater extent.     

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...     

Elongating RNA polymerase II is disassembled through specific degradation of its largest but not other subunits in response to DNA damage in vivo

Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.     

The bacteriophage T4 regulatory protein gpunf/alc binds to DNA in the absence of RNA polymerase.

DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf/alc. The unf/alc protein does not bind to DNA via an association with RNA polymerase; gpunf/alc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.