Duplication of single stranded DNA catalyzed by calf thymus DNA polymerase alpha.

Purified calf thymus DNA polymerase alpha is inactive with native DNA as template and shows little activity with denatured DNA. DNA synthesis with denatured DNA as template is greatly stimulated by the addition of a nuclease which initially copurifies with DNA polymerase but is separated from the polymerase on DEAE-cellulose chromatography. A limit digest of nuclease treated native DNA which is then denatured is replicated 80-95%; extensive replication is also obtained with native DNA partially degraded by pancreatic DNase and then denatured. The product of the reaction with calf thymus nuclease-treated DNA as template is double-stranded DNA with a hairpin (looped back) structure.     

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.     

Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Denaturation of deoxyribonucleic acid in situ effect of formaldehyde.

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.     

Recognition of cisplatin-DNA interstrand cross-links by replication protein A

Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.     

Characterization of Bleomycin-Resistant DNA

After reaction of DNA with high concentrations of bleomycin, approximately 80% of the DNA becomes trichloroacetic acid (TCA) soluble. The remaining 20% of the DNA remains TCA insoluble. Upon further treatment of this TCA-insoluble material with high concentrations of the drug, no further drug action can be detected. Drug action is defined as fragmentation of DNA to smaller molecular size, release of free bases, and TCA solubilization. This material which is not attacked by bleomycin has been termed bleomycin-resistant DNA. This bleomycin-resistant DNA does not compete with native DNA in the bleomycin reaction indicating that there is no binding or inactivation of the drug by the resistant DNA. The resistant DNA shows very little hyperchromicity when heated through the melting temperature for the corresponding native DNA, indicating a single-stranded structure. Results of sedimentation and equilibrium analyses yield a molecular weight of about 4,000 daltons. This value is the same regardless of the source of the native DNA. Finally, the bleomycin-resistant DNA exhibits a base composition similar to that of the native DNA from which it was derived.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates.

We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication.     

Interspersion of mouse satellite deoxyribonucleic acid sequences

DNA sequences with homology to the major (A + T)-rich mouse satellite component were localized in CsCl gradients by hybridization with a labeled satellite cRNA probe. Although, as expected, most of the hybridization was to DNA in the satellite-rich shoulder, substantial radioactive cRNA hybridized with DNA from denser regions of the gradient. Further examination revealed that hybridization to main-band DNA was not due to physical trapping of satellite DNA in the gradient, and melting experiments argue that the associated radioactivity was due to true RNA/DNA hybridization. Nearest-neighbor analysis of hybridized [alpha-32P]CTP-labeled l-strand cRNA indicates that hybridization to main-band DNA is by the satellite cRNA and not a contaminant. Together, these data argue that mouse satellite-like sequences are interspersed within the main-band fraction of DNA. For the support of this contention, total mouse DNA, purified main-band DNA, and purified satellite DNA were digested with EcoRI, sedimented in a sucrose gradient, and hybridized with labeled satellite cRNA. Mouse satellite DNA is not cleaved with EcoRI, so that purified EcoRI-digested satellite DNA sediments as a high molecular weight component. When total mouse DNA is digested with EcoRI, the majority of satellite-like sequences remain as high molecular weight DNA; however, significant amounts of satellite-like sequences sediment with the bulk of the lower molecular weight digested DNA, lending further credence to the argument that satellite-like sequences are interspersed with main-band DNA.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli

The pairing of single- and double-stranded DNA molecules at homologous sequences promoted by recA and single-stranded DNA-binding proteins of Escherichia coli follows apparent first-order kinetics. The initial rate and first-order rate constant for the reaction are maximal at approximately 1 recA protein/3 and 1 single-stranded DNA-binding protein/8 nucleotides of single-stranded DNA. The initial rate increases with the concentration of duplex DNA; however, the rate constant is independent of duplex DNA concentration. Both the rate constant and extent of reaction increase linearly with increasing length of duplex DNA over the range 366 to 8623 base pairs. In contrast, the rate constant is independent of the size of the circular single-stranded DNA between 6,400 and 10,100 nucleotides. No significant effect on reaction rate is observed when a single-stranded DNA is paired with 477 base pairs of homologous duplex DNA joined to increasing lengths of heterologous DNA (627-2,367 base pairs). Similarly, heterologous T7 DNA has no effect on the rate of pairing. These findings support a mechanism in which a recA protein-single-stranded DNA complex interacts with the duplex DNA to produce an intermediate in which the two DNA molecules are aligned at homologous sequences. Conversion of the intermediate to a paranemic joint then occurs in a rate-determining unimolecular process.     

Studies on the fate of homologous DNA applied to seedlings of Matthiola incana.

Seedlings of Matthiola incana (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA. Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants. When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form. The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments. Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.     

Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.

DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.     

Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase

BACKGROUND: DNA primases catalyse the synthesis of the short RNA primers that are required for DNA replication by DNA polymerases. Primases comprise three functional domains: a zinc-binding domain that is responsible for template recognition, a polymerase domain, and a domain that interacts with the replicative helicase, DnaB. RESULTS: We present the crystal structure of the zinc-binding domain of DNA primase from Bacillus stearothermophilus, determined at 1.7 A resolution. This is the first high-resolution structural information about any DNA primase. A model is discussed for the interaction of this domain with the single-stranded DNA template. CONCLUSIONS: The structure of the DNA primase zinc-binding domain confirms that the protein belongs to the zinc ribbon subfamily. Structural comparison with other nucleic acid binding proteins suggests that the beta sheet of primase is likely to be the DNA-binding surface, with conserved residues on this surface being involved in the binding and recognition of DNA.     

Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.     

Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.     

Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.     

Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction

DNA molecules carrying a Mu end(s) are inefficient targets in the Mu DNA strand-transfer reaction. This target immunity is due to preferential dissociation of Mu B protein from DNA molecules that have Mu A protein bound to the Mu end; free DNA is a much poorer target than DNA with Mu B protein bound. We show that Mu B protein, which binds nonspecifically to DNA, is immobile once bound. An encounter between Mu A and Mu B proteins, bound some distance apart along DNA, is necessary to facilitate the Mu B dissociation. Experiments which show that DNA without a Mu end can acquire immunity, by catenation to DNA with a Mu end(s), are consistent with a model of Mu A-Mu B interaction by DNA looping, but not by linear movement of protein(s) along DNA.