In Situ Activity of Enzymes on Polyacrylamide Gels of a Deoxyribonucleic Acid-Membrane Fraction Extracted from Pneumococci

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.     

Phagostimulation of larval Culexpipiens L. by nucleic acid nucleotides, nucleosides and bases

ABSTRACT. The influence of nucleic acid constituents on the rate of ingestion of charcoal powder filtered from ambient water by larvae of Culex pipiens L. was examined. All nucleotides tested stimulated ingestion to some extent. Various mono-, di- and tri-phosphates of adenosine were most effective and at concentrations of 1 mM stimulated ingestion nearly as well as yeast extract, a powerful phagostimu-lant. Guanylic, thymidylic, cytidylic and uridylic acids were less stimulatory, the latter two even at 10 mM. Cyclic AMP and deoxyadenylic acid were less effective than other adenine nucleotides. The nucleosides adenosine, guanosine and uridine were almost as effective as their corresponding nucleotides (adenylic, guanylic and uridylic acids); thymidine was less effective than thymidylic acids, whereas cytidine was non-stimulatory. Adenine, guanine, uracil and cytosine, the bases of the ribose nucleotides, were non-phagostimulatory, whereas thymine, base of the deoxynucleotide, thymidylic acid, caused low but significantly increased ingestion. These findings are compared with the reported phagostimulation by nucleic acid constituents of certain plant feeding insects and with the stimulation of engorgement of the blood meal by many blood feeding insects.     

Thymidylic Acid Synthesis in Eimeria tenella (Coccidia) Cultured In Vitro*

The source of thymidylic acid for DNA synthesis in 1st generation stages of Eimeria tenella cultured in vitro was investigated using 5-bromo-2′-deoxyuridine, a thymidine analog, and 5-fluoro-2′-deoxyuridine, an inhibitor of de novo thymidylic acid synthesis. Results show that the parasite is unable to utilize either exogenous thymidine or 5-bromo-2′-deoxyuridine but is dependent upon the methylation of deoxyuridylic acid via the thymidylate synthetase reaction to supply thymidine for DNA synthesis.     

Kinetics of methylation in Escherichia coli K-12.

Newly synthesized DNA is undermethylated in E. coli K-12. The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations.     

Cloning of rat alpha-fetoprotein 3'-terminal complementary deoxyribonucleic acid sequences and preparation of radioactively labeled hybridization probes from cloned deoxyribonucleic acid inserts

Double-stranded complementary deoxyribonucleic acid (cDNA) was synthesized from rat yolk sac alpha-fetoprotein (AFP) mRNA, inserted into the PstI site of plasmid pBR322 by an oligo(deoxyguanylic acid).oligo(deoxycytidylic acid) joining technique, and cloned in Escherichia coli chi 1776. A plasmid containing an inserted AFP double-stranded cDNA with a contiguous poly(adenylic acid) [poly(A)] segment was identified and subsequently employed in a new method for preparing AFP-specific hybridization probe. Following an initial digestion of the AFP plasmid with HindIII to create an open, recessed 3' end, lambda exonuclease III was employed to remove the DNA strand opposite the coding strand of the cDNA insert. Oligo(thymidylic acid) was then annealed to the poly(A) segment and employed as primer for E. coli DNA polymerase I to synthesize a 32P-labeled cDNA copy of the AFP coding strand. The single-stranded cDNA product was easily isolated by sedimentation through isokinetic alkaline sucrose gradients. Hybridization with this AFP-specific cDNA probe showed that the yolk sac contained a 6-fold greater concentration of AFP mRNA than that of the fetal liver. AFP mRNA was also found in the normal adult liver, but at a much lower level than in the fetal liver. The concentrations of AFP mRNA in Morris hepatomas 7777 and 8994, however, were significantly elevated to a 2- to 3-fold higher concentration that in the fetal liver.     

Dietary nucleotide requirements of the mosquito, Culex pipiens

Dietary nucleic acid, or certain constituents thereof, is essential for full development of the mosquito Culex pipiens. Yeast RNA, whole or hydrolysed, is fully effective. Sperm DNA is totally ineffective on its own, but becomes fully effective when supplemented with uridylic acid. RNA can be replaced with only partial success by a mixture of its component nucleotides (adenylic, guanylic, cytidylic, and uridylic acids), but with the addition of thymidylic acid (characteristic of DNA but present in RNA only as a minor component of transfer RNA), the resulting five-nucleotide mixture is a fully adequate replacement for yeast RNA. Single and multiple deletions from this five-nucleotide mixture showed a minimal requirement for three nucleotides: adenylic acid (a purine ribonucleotide) and thymidylic acid (a pyrimidine deoxyribonucleotide) were both specifically required; as the third nucleotide, either cytidylic or uridylic acid (both pyrimidine ribonucleotides) was equally satisfactory. Mixtures of the five nucleosides or five deoxynucleotides corresponding in base composition to the effective five-nucleotide mixture were only partially effective substitutes.     

Iododeoxyuridine administered to mice is de-iodinated and incorporated into DNA primarily as thymidylate.

Iododeoxyuridylic acid, a structural analog of thymidylic acid, is extensively de-iodinated in vivo by the enzyme thymidylate synthetase. Substantial amounts of the deoxyuridylic acid formed by this process are subsequently methylated and incorporated into DNA as thymidine. As a result, when mice are given tritiated iododeoxyuridine, most of the tritium incorporated into their DNA is present in thymidine rather than in iododeoxyuridine. Some, but not nearly as much, tritium from tritiated bromodeoxyuridine is also incorporated into DNA thymidine.     

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

Synthesis and nucleic acids binding properties of diastereomeric aminoethylprolyl peptide nucleic acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the "cis-" homothymine aepPNA decamers with (2'R,4'R) and (2'S,4'S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2'R,4'S) and (2'S,4'R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2'R,4'R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

DNase VII of human placenta. Mechanism studies

The mechanism of the human placental DNase VII, described previously (Hollis, G. F., and Grossman, L. (1981) J. Biol. Chem. 256, 8074-8079) has been investigated in further detail. The enzyme initiates exonucleolytic hydrolysis from the 3'-end of DNA in a nonprocessive, or distributive, manner, regardless of whether the carbohydrate moiety associated with the 3'-terminal nucleotide contains H or OH at its 2' and 3' positions. DNase VII does not have associated RNase H activity; however, it is capable of removing 3'-terminal ribonucleotides. The enzyme also can hydrolyze DNA containing a terminal nucleotide lacking a purine or pyrimidine as well as termini containing noncomplementary nucleotides. DNase VII activity is product-inhibited by deoxynucleoside 5'-monophosphates. From kinetic studies, the mononucleotide deoxyadenylic acid is a noncompetitive inhibitor with a Ki = 0.3 mM. The resemblance of DNase VII to the 3'----5' exonuclease activity of Escherichia coli DNA polymerase I and its possible role in excision repair and proofreading are discussed.     

Studies on a thermophilic RNA polymerase which is active only on poly d(A-T) and poly dAdT.

Two types of RNA polymerases [EC 2.7.7.6], polymerases A and B, exist in thermophilic bacteria, Thermus thermophilus HB8. Polymerase B is apparently like the core enzyme of polymerase A but is active only when an alternating copolymer of deoxyadenylic and deoxythymidylic acids (poly d(A-T)) or a mixture of homopolymers of deoxyadenylic acid and deoxythymidylic acid (poly dAdT) is used as a template. Polymerase B was further characterized to elucidate its relation to polymerase A and to determine why it is inactive on natural DNA's. 1. Polymerase B did not show pyrophosphate exchange activity. Dinucleoside monophosphates did not activate the RNA-synthesizing activity. The results suggested that polymerase B had no initiation and presumably no elongation activities. 2. Polymerase B had about 6 times greater affinity to DNA than polymerase A. The binding of polymerase B to DNA was, however, reversible. The complex of DNA with polymerase A was stable and the polymerase was not removed from the initial complex even when a large amount of DNA was added. 3. E. coli sigma subunit could not stimulate the activity of polymerase B toward DNA's. 4. Polymerase B could utilize poly d(A-T) and poly dAdT as templates, but could not use Bacillus cereus DNA though the structure is reported to be similar to that of poly d(A-T).     

Free radical mechanisms in neocarzinostatin-induced DNA damage

The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.     

DNA polymerases that propagate the eukaryotic DNA replication fork

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.     

Quantitative reactions of anti 5,9-dimethylchrysene dihydrodiol epoxide with DNA and deoxyribonucleotides

Native as well as denatured calf thymus DNA, deoxyguanylic and deoxyadenylic acid, respectively, were reacted with the racemic anti 5,9-dimethylchrysene dihydrodiol epoxide (5,9-DMCDE). The deoxyribonucleoside adducts were separated by HPLC and characterized by CD and NMR. Approximately 17% of the epoxide was trapped by native DNA and 76% of the adducts were derived from the RSSR enantiomer. The ratios of dAdo/dGuo modification in DNA were 14/86 and 19/81 for RSSR and SRRS enantiomers, respectively. By monitoring the product yields of anti 5,9-DMCDE with DNA and deoxyribonucleotides, we hoped to gain further insight into the factors responsible for deoxyguanosine adduct formation by 5-methylchrysene dihydrodiol epoxide (5-MCDE) compared to 5, 6-dimethylchrysene dihydrodiol epoxide (5,6-DMCDE). The adduct yields in deoxyribonucleotide reactions of 5,9-DMCDE were slightly higher than those from 5-MCDE. However, the reaction yields of 5, 9-DMCDE with DNA were lower than those with 5-MCDE in most cases, particularly for the cis and trans deoxyadenosine adducts. It seems that the 9-methyl group of 5,9-DMCDE significantly influences adduct formation with the deoxyadenosine residue in DNA in contrast to the 6-methyl group of 5,6-DMCDE. The 9-methyl group sterically decreases deoxyadenosine adduct yields more in reaction with native DNA than denatured DNA, but it has little effect on deoxyribonucleotide reactions. Adduct formation with deoxyguanosine residues in DNA by all three dihydrodiol epoxides correlate with their respective tumorigenic and mutagenic activities.     

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase δ, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.     

Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease

The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins. We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction. Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles. Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end. ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases. Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end. In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8. This study further illustrates the complexity of the multi-functional ICP8 protein.     

Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.     

Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.     

Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.