Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.     

Sequence-specific interaction of sigma subunit of E. coli RNA polymerase with DNA.

The interaction of sigma subunit of E. coli RNA polymerase with DNA, either double or single-stranded, and with two inhibitors of RNA synthesis was investigated by using antibodies directed against the subunit. Free sigma subunit was shown to interact with poly(dA), poly(dT), poly(dAC).poly(dGT), T7 DNA and, to a lesser degree, with lambda DNA. When the sigma subunit forms part of the holo enzyme, sigma also interacts with poly(dG).poly(dC). Rifampicin and streptolydigin interact with sigma in the holo enzyme and with free and core bound sigma subunit, respectively. The results suggest that sigma recognizes mainly AC-GT-sequences in double-stranded DNA. The findings are correlated with the base composition in RNA polymerase binding regions of promoters and suggest at least a general interaction between sigma subunit and single-stranded DNA in open complexes.     

Ribonuclease H: a Ubiquitous Activity in Virions of Ribonucleic Acid Tumor Viruses

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.     

RNA polymerase: linear competitive inhibition by bis-(3' to 5')-cyclic dinucleotides,NpNp.

We have investigated the possible role of the bis-(3' to 5')-cyclic dinucleotides UpUp and ApUp as kinetic inhibitors of the DNA dependent RNA polymerase enzyme of E. coli, using T7 delta D111 deletion mutant DNA and several synthetic DNA polymers as templates. We have established that UpUp is a linear competitive inhibitor of the initiation phase of the polymerization (Ki = 28 microM using T7 delta D111 DNA as a template), but that it has no effect when added during the elongation phase. The compound ApUp is an inhibitor of the reaction only when poly(dA-T).poly(dA-T) is used as a template, and UpUp is an inhibitor of the reaction when poly(dA).poly(dT) was employed as the DNA template.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Purification and partial characterization of megamodulin, a heat-stable protein factor from E. coli and its stimulatory effect on RNA polymerase holoenzyme

Megamodulin, a heat-stable protein from Escherichia coli was isolated and purified near homogeneity as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. It had a molecular weight of 71,000 and pl between 3.5 and 4.0. This factor stimulated E. coli RNA polymerase 71-fold in the presence of a synthetic template such as poly (rA).p(dT). When TATAAA sequence was used as template, the RNA polymerase activity was increased 68 times by this factor. The possible mechanism by which this protein factor may regulate the RNA polymerase activity has been described.     

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.     

The incorporation of wrong bases by DNA polymerase I following gamma-irradiation of DNA-like templates

The synthesis of polydeoxyribose polymers by Escherichia coli DNA polymerase I has been investigated with control and gamma-irradiated DNA-like polymer templates containing only two bases. The results show that irradiation of a poly(dA) strand leads to the incorporation of dG, whereas irradiation of poly(dC) and poly(dG) strands both lead to the incorporation of dA. Irradiation of poly(dT) does not lead to the incorporation of any wrong base. The wrong bases are incorporated into the complementary strand of the newly synthesised DNA.     

Single-strand binding proteins from phage T4 and E. coli form higher order structures with poly(dT)

Complexes of poly(dT) with gene 32 protein from phage T4 or E. coli single-strand binding protein were digested by nuclease P1 from Penicillum citrinum. Protected fragments were analyzed by gel electrophoresis. In both cases, a series of bands was obtained corresponding to multiples of a repeat unit whose size was about 80 nucleotides. Such protected fragments could not be detected under the same experimental conditions when poly(dA) was used instead of poly(dT). The formation of nucleosome-like structures is discussed in relation to the higher affinity exhibited by single-strand binding proteins towards poly(dT).     

The DNA sequence specificity of stimulation of DNA polymerases by factor D

The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.     

Purification and properties of a light-inducible nuclease from Euglena gracilis.

The nuclease described by Carell, E.F., Egan, J.M. and Pratt, E.A. [Arch. Biochem. Biophys. (1970) 138, 26-31] has been purified 1000-fold from Euglena gracilis strain Z. The enzyme catalyzes the hydrolysis of both polyribonucleotides and polydeoxyribonucleotides. The relative rates of hydrolysis of synthetic and natural polynucleotides was found to be: poly (U) 100, poly (dT) 33, denatured calf-thymus DNA 33, yeast tRNA 9, E. coli total RNA 6, poly (dA dT) 5, poly (A) less than 1, poly (C) less than .05, and poly (G) less than .05. The enzyme attacks polynucleotides in an endonucleolytic fashion, yielding products terminated with a 3'-phosphate. Poly (U) appears to be hydrolyzed completely to 3'-UMP; both RNA and DNA appear to have some phosphodiester bonds resistant to enzyme catalyzed hydrolysis. Because of its mode of action and its inducibility by light, we propose the name endonuclease L for this enzyme.