Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes

Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-³²P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.     

The binding of topoisomerase I to T antigen enhances the synthesis of RNA-DNA primers during simian virus 40 DNA replication

Topoisomerase I (topo I) is required for the proper initiation of simian virus 40 (SV40) DNA replication. This enzyme binds to SV40 large T antigen at two places, close to the N-terminal end and near the C-terminal end of the helicase domain. We have recently demonstrated that the binding of topo I to the C-terminal site is necessary for the stimulation of DNA synthesis by topo I and for the formation of normal amounts of completed daughter molecules. In this study, we investigated the mechanism by which this stimulation occurs. Contrary to our expectation that the binding of topo I to this region of T antigen provides the proper unwound DNA substrate for initiation to occur, we demonstrate that binding of topo I stimulates polymerase alpha/primase (pol/prim) to synthesize larger amounts of primers consisting of short RNA and about 30 nucleotides of DNA. Topo I binding also stimulates the production of large molecular weight DNA by pol/prim. Mutant T antigens that fail to bind topo I normally do not participate in the synthesis of expected amounts of primers or large molecular weight DNAs indicating that the association of topo I with the C-terminal binding site on T antigen is required for these activities. It is also shown that topo I has the ability to bind to human RPA directly, suggesting that the stimulation of pol/prim activity may be mediated in part through RPA in the DNA synthesis initiation complex.     

The primase activity of DNA polymerase alpha from calf thymus

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.     

Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or lambda deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPO1, or phi29 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of gamma-(32)P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and lambda DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or lambda DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.     

Monoclonal antibodies to DNA modified by 8-methoxypsoralen and ultraviolet A light.

A panel of monoclonal antibodies have been developed which specifically recognize DNA modified by 8-methoxypsoralen (8-MOP) and ultraviolet A light (320-400 nm) (UVA). These antibodies have been characterized as to sensitivity and specificity by an enzyme linked immunosorbent assay (ELISA). In a competitive ELISA with the most sensitive antibody, 50% inhibition of antibody binding occurred at 17 fmole 8-MOP-DNA photo adducts. One adduct per 10(7) bases could be reliably detected. There was also some antibody cross-reactivity with DNAs modified by 4' aminomethyl-4, 5, 8-trimethylpsoralen and 4', 5-dimethylangelicin as well as DNA isolated from cells treated with 8-MOP and UVA. The primary specificity of one of the antibodies was shown to be the 4', 5' thymine monoadduct by competitive inhibition studies using HPLC fractions of an enzymatic digest of 8-MOP poly(dA-dT) . poly(dA-dT). These antibodies should allow the quantitation of adduct levels in various in vitro systems as well as humans exposed clinically to 8-MOP and UVA.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

Antibacterial nitroacridine, Nitroakridin 3582: binding to nucleic acids in vitro and effects on selected cell-free model systems of macromolecular biosynthesis

Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes.     

Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3' cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3' single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3'-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

T4 DNA polymerase: Rates and processivity on single-stranded DNA templates

Three different methods have been used to determine the rate at which an individual bacteriophage T4 DNA polymerase molecule moves when synthesizing DNA on a single-stranded DNA template chain. These methods agree in suggesting an in vitro rate for this enzyme of about 250 nucleotides per second at 37 °C. This rate is close to the rate at which bacteriophage T4 replication forks move in vivo (about 500 nucleotides per second). Comparison with the overall amount of DNA synthesis seen in in vitro reactions reveals that only a small fraction of the T4 DNA polymerase molecules present are synthesizing DNA at any one time. This is explicable in terms of the limited processivity of the enzyme in these reactions, along with its capacity for non-productive DNA binding to the DNA template molecules.