Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Modified polynucleotides. I. Investigation of the enzymatic polymerization of 5-alkyl-dUTP-s.

The chemical synthesis of 5-alkyl-dUTP-s and their participation as substrates in poly[d(A-6)] primed polymerization reactions with dATP by E. coli DNA polymerase I enzyme has been described. In comparison with dTTP, at saturating substrate concentrations, the rate of hypochromic effect was found to be 17.3% higher for dUTP and was lower by 27.4% for 5-ethyl-dUTP, 29.5% for 5-n-propyl-dUTP, 31.4% for 5-n-butyl-dUTP and by 85.0% for 5-n-pentyl-dUTP. No hypochromic effect could be observed, however, with 5-iso-propyl-, 5-tert.butyl- and 5-n-hexyl-dUTP-s. Polydeoxynucleotides have also been isolated from the reaction mixture and some of their structural properties determined.     

Processiveness of DNA polymerases. A comparative study using a simple procedure.

In this communication, we describe a simple procedure for analyzing the processiveness of DNA polymerases in general. By choosing conditions for which the number of incorporations per available primer is less than 1, we have reduced the probability of a primer molecule being utilized by the enzyme more than once. The primer-template used was poly(dA)300:oligo(dT)10, and the product was isolated by oligo(dT)-cellulose chromatography. The number of dTMP residues added per association was determined from the [3H]dThd + [3'-3H]dTMP/[3H]dThd ratio of the product after its digestion by micrococcal nuclease and spleen phosphodiesterase. Using this procedure, we have found that Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus alpha- and beta-DNA polymerase are "quasi-processive." Most of these enzymes add on the average approximately 10 to 15 nucleotides before dissociating from the template. T5 DNA polymerase, on the other hand, is processive, i.e. it continues to replicate a given template until it is very close to the 5' end of the template. With "nicked DNA-like" poly(dA):oligo(dT), the processiveness of E. coli DNA polymerase I is increased 2- to 2.5-fold. The significance of this increase in determining the "patch size" during DNA repair is discussed.     

RNA polymerase. Direct evidence for a unique topographical site for initiation

The compound 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl)adenine 5'-monophosphate is an inhibitor (Ki = 330 microM) of the initiation binding site of the DNA-dependent RNA polymerase derived from Escherichia coli. The alpha-32P derivative of this photo-labile compound is used to derivatize a site on the sigma subunit of the holoenzyme (E sigma) using either T7 delta D111 or poly[d(A-T)] as a DNA template. The incorporation of the 32P label into the sigma subunit could be prevented by the addition of either 5'-AMP or 5'-ATP. The results are suggested to support the existence of a unique initiation binding site, topographically distinct from the sites employed during the elongation phase.     

Modified polynucleotides. VI. Properties of a synthetic DNA containing the anti-herpes agent (E)-5-(2-bromovinyl)-2''-deoxyuridine

A new modified polydeoxynucleotide, a copolymer of nucleotides of 2'-deoxyadenosine and the very efficacious anti-herpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine was synthesized with E. coli DNA polymerase I enzyme. It is characterized by its physical (absorption and circular dichroism spectra, thermal transition, sedimentation analysis) and bioorganic (template activity, stability) properties. Compared to poly [d(A-T)], the modified polydeoxynucleotide had a lower thermal stability but exhibited higher stability against DNases and higher template activity for DNA synthesis. Template activity for RNA synthesis of this template was, however, poor and extent of AMP and UMP incorporation was limited as well.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.     

polA6, A mutation affecting the DNA binding capacity of DNA polymerase I.

The polA6 mutation is an allele of the polA gene of Escherichia coli which produces a DNA polymerase I species readily distinguishable from that produced by the wild type allele. Experiments described here show that this enzyme has an altered pH optimum for polymerization and a lower binding affinity for DNA. The defect clearly lies within the carboxyl-terminal large fragment of the enzyme produced by in vivo or in vitro proteolysis since the fragment has the same pH optimum for polymerization as the intact enzyme. The polA6 enzyme and its fragment are more sensitive to phosphate ions than the wild type polymerase, and the large fragment is less efficient at binding poly d(AT) in in vitro binding assays. Although the specific nucleolytic activity of the polA6 enzyme is higher than that of the wild type, there is no apparent alteration in pH optimum for the hydrolysis of eigher double or single stranded DNA.     

Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.     

Excision in vitro of the DNA bound carcinogen, 4-nitroquinoline 1-oxide.

It has been shown that 4-hydroxyaminoquinoline 1-oxide, the proximate form of the carcinogen 4-nitroquinoline 1-oxide, binds covalently to the purine bases of DNA. Here we report that carcinogen-bound nucleotides can be excised from DNA by a 5' leads to 3' exonuclease associated with DNA polymerase I of E. coli in the forms of either mononucleotides or oligonucleotides. Beef spleen phosphodiesterase II (5' leads to 3') also split carcinogen-bound nucleotides, while a 3' leads to 5' exonuclease of DNA polymerase I and E. coli exonuclease III (3' leads to 5') could not excise the modified nucleotide.