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PubMed1998

Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine.

The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.

Amino Acid Sequence↗

PubMed1989

Effects of chloroquine on the torsional dynamics and rigidities of linear and supercoiled DNAs at low ionic strength.

The magnitude and uniformity of the torsion elastic constant (alpha) of linear and supercoiled pBR322 DNAs are measured in 3 mM Tris as a function of added chloroquine/basepair ratio (chl/bp) by studying the fluorescence polarization anisotropy of intercalated ethidium dye. The time-resolved FPA is measured using a picosecond dye-laser for excitation and time-correlated single-photon counting detection. For both linear and supercoiled DNAs, alpha remains uniform except at the very highest chl/bp ratio examined. For the linear DNA, alpha decreases from 5.0 x 10(-12) dyne-cm at chl/bp = 0 to about 3.5 x 10(-12) dyne-cm at chl/bp = 0.5, and remains at that value up to chl/bp = 5, whereupon it increases back up to its original value. For the supercoiled DNA, alpha remains constant at about 5.2 x 10(-12) dyne-cm from chl/bp = 0 up to chl/bp = 5, whereupon it increases in parallel with the linear DNA. The effect of chloroquine on the secondary structure, torsion constant, and torsional dynamics evidently differs substantially between linear and supercoiled DNAs, even under conditions where the supercoiled DNA is completely relaxed and both DNAs bind the same amount of dye. This strongly contradicts any notion that the local structures of linear and relaxed supercoiled DNA/dye complexes with the same binding ratio are identical. The increase in apparent alpha at chl/bp = 5 for both DNAs may be due to stacking of the chloroquine in the major groove and consequent stiffening of the filament.

Chloroquine↗

PubMed1999

Targeted linearization of DNA in vivo.

In the past decade, site-specific chromosomal DNA cleavage mediated by DNA endonucleases has been used to examine diverse aspects of chromosome structure and function in eukaryotes, such as DNA topology, replication, transcription, recombination, and repair. Here we describe a method with which chromosomes can be linearized at any predefined position in vivo. Yeast homothallic switching endonuclease (HO endo), a sequence-specific double-strand nuclease involved in mating-type switching, is employed for targeting DNA cleavage. HO endo contains discrete functional domains: a N-terminal nuclease and a C-terminal DNA-binding domain, thereby allowing construction of a chimeric nuclease with the cutting site distinct from the original HO recognition sequence. The expression of the nuclease is engineered to be controlled by a tightly regulated, inducible promoter. The cut sites recognized by HO endo or its derivatives are introduced specifically at desired positions in the yeast genome by homologous recombination. Here we present experimental procedures and review some applications based on this approach in yeast and other biological systems.

Blotting, Southern↗

PubMed1998

Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2.

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins are in vitro substrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.

Amino Acid Sequence↗

PubMed1986

Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.

Chromosome Mapping↗

PubMed1985

An equilibrium between distorted and undistorted DNA in the adult chicken beta A-globin gene.

We have used single strand specific nucleases to map DNA distortion in the adult chicken beta A-globin gene. We have detected two structures of that kind and have mapped nuclease-cutting sites at one base resolution. One prominent site is centered at -190 relative to the RNA capping site and is positioned at the center of a stretch of contiguous C residues. The second site is near the first intron/exon junction (+620) and appears as a series of discrete 1-base-long enzyme-cutting sites. Based upon the pattern of nuclease cutting and the kinetics of nuclease cutting we conclude that the "poly(C)" stretch may assume a looped geometry in supertwisted DNA molecules which is similar to that proposed by Felsenfeld (Nickol, J. M., and Felsenfeld, G. (1983) Cell 35, 467-477). We show that S1 nuclease cuts within the intron occur mainly at the end points of polypurine segments and suggest that such end points may assume a distorted transitional geometry. We find that Neurospora crassa endonuclease cuts both the promotor and intron sites in linear DNA molecules but that in linear DNA the cutting process is limited by a first order conformation change of the DNA substrate. Based upon those kinetics we propose that in unstressed DNA, each of the two sites can convert between a distorted and undistorted geometry. In the enzyme assay buffer at 37 degrees C, the time constant for the equilibrium is nearly 10 h for the promotor site and 7 h for the intron.

Animals↗

PubMed1998

Comparison of the formation of 8-hydroxy-2'-deoxyguanosine and single- and double-strand breaks in DNA mediated by fenton reactions.

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1994

Distance and end configuration effects on VDJP-mediated DNA joining.

We have previously reported the cloning of a protein, VDJP, that is capable of binding the nonamer element of the V(D)J Recombinational Signal Sequence (RSS) as well as joining linear DNA fragments containing RSS elements in vitro. We show here that the linearized DNA molecules must contain a 5' extension or blunt end in order to be joined by VDJP. DNAs with 3' extensions are not efficiently joined by VDJP. Furthermore, the joining activity of DNAs with 5' extensions is significantly increased as the distance between the end and the RSS decreases. It is not yet clear what role VDJP plays in vivo, because our assay may not mimic exactly the in vivo DNA intermediates.

Animals↗

PubMed1994

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

Bacterial Proteins↗

PubMed1984

Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites.

The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP. Furthermore, Cre is able to mediate a synaptic union of two loxP sites. When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex. These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination.

Base Sequence↗

PubMed1992

Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.

Antigens, Nuclear↗

PubMed1997

Initiation of bacteriophage phi29 DNA replication in vivo: assembly of a membrane-associated multiprotein complex.

Initiation of in vitro phage phi29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase. We have analyzed membrane vesicles from phi29-infected Bacillus subtilis cells by quantitative immunoblot techniques. During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase. Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked. Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA. The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted. The above results, together with the critical role of protein p1 for in vivo phi29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo phi29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase. Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP. The ability of free TP to bind to membranes and to prime phi29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins. We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane.

Bacillus Phages↗

PubMed1991

Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity.

Calcium↗

PubMed2005

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase.

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.

Adenosine Triphosphatases↗

PubMed1993

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule.

The SLP2 plasmid had previously been demonstrated genetically to exist in Streptomyces lividans by its ability to promote conjugation and to elicit 'pocks' on recipient (SLP2-) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50 kb linear DNA was isolated from SLP2+ but not SLP2- strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44bp imperfect terminal inverted repeat. The terminal 12bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by lambda exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a free form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.

Base Sequence↗

PubMed2006

Protein-induced DNA bending clarifies the architectural organization of the sigma54-dependent glnAp2 promoter.

Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.

Base Sequence↗

PubMed1992

Analysis of the defect in DNA end joining in the murine scid mutation.

Murine severe combined immune deficiency (scid) is marked by a 5,000-fold reduction in coding joint formation in V(D)J recombination of antigen receptors. Others have demonstrated a sensitivity to double-strand breaks generated by ionizing radiation and bleomycin. We were interested in establishing the extent of the defect in intramolecular and intermolecular DNA end joining in lymphoid and nonlymphoid cells from scid mice. We conducted a series of studies probing the ability of these cells to resolve free ends of linear DNA molecules having various biochemical end configurations. We find that the stable integration of linear DNA into scid fibroblasts is reduced 11- to 75-fold compared with that in normal fibroblasts. In contrast, intramolecular and intermolecular end joining occur at normal frequencies in scid lymphocytes and fibroblasts. This normal level of end joining is observed regardless of the type of overhang and regardless of the requirement for nucleolytic activities prior to ligation. The fact that free ends having a wide variety of end configurations are recircularized normally in scid cells rules out certain models for the defect in scid. We discuss the types of DNA end joining reactions that are and are not affected in this double-strand break repair defect in the context of a hairpin model for V(D)J recombination.

Animals↗

PubMed1983

Characterisation of cauliflower mosaic virus DNA forms isolated from infected turnip leaves.

Several different forms of cauliflower mosaic virus (CaMV) DNA were detected in nucleic acid preparations from CaMV-infected turnip leaves. As well as supercoiled and open-circular molecules, various linear DNA structures were identified. The relative amounts of these DNA forms varied in plants infected with different CaMV isolates. Restriction enzyme mapping and one- and two-dimensional gel electrophoresis revealed the presence of linear molecules apparently formed by breaks in the second strand at each of the three discontinuities. Two major linear DNA forms are double-stranded over part of their length and appear to have single-stranded extensions of the -strand of variable length. Since these DNA forms are not produced during extraction and probably exist as unencapsidated or partially encapsidated molecules, they may represent intermediates either in DNA replication or in virion assembly.

DNA Restriction Enzymes↗