Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate–nitric oxide neurotransmission

Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD+ to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD+ and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1−/−), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving N-methyl-d-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS−/−). An increase in NAD+ levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS−/− mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.

A nicked duplex decamer DNA with a PEG6 tether

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure 1H, 31P and T1 spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG6 linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The 1H spectra also show much sharper resonances in the presence of K+ indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the 1H resonances have longer T1 times. This parameter is suggested as a sensitive gauge of stabilisation.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery

The product of the herpes simplex virus type 1 UL28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of UL28 in this process, purified UL28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for UL28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by UL28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by UL28 protein. To determine the relevance of the observed UL28 protein–pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of UL28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by UL28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the UL28 protein–pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the UL28-encoded component of the herpesvirus cleavage and packaging machinery.

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5′-3′ and the other with 3′-5′ polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3′ ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative “bubble migration” mode of RDR could play a role in lesion bypass by the T4 replication machinery.

Tuning DNA “strings”: Modulating the rate of DNA replication with mechanical tension

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme–template complex indicate n = 1. We also present a model for the “tuning” of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

Inter-strand C-H...O hydrogen bonds stabilizing four-stranded intercalated molecules: stereoelectronic effects of O4' in cytosine-rich DNA.

DNA fragments with stretches of cytosine residues can fold into four-stranded structures in which two parallel duplexes, held together by hemiprotonated cytosine.cytosine+ (C.C+) base pairs, intercalate into each other with opposite polarity. The structural details of this intercalated DNA quadruplex have been assessed by solution NMR and single crystal x-ray diffraction studies of cytosine-rich sequences, including those present in metazoan telomeres. A conserved feature of these structures is the absence of stabilizing stacking interactions between the aromatic ring systems of adjacent C.C+ base pairs from intercalated duplexes. Effective stacking involves only the exocyclic keto groups and amino groups of the cytidine bases. The apparent absence of stability provided by stacking interactions between the bases in this intercalated DNA has prompted us to examine the available structures in detail, in particular with regard to unusual features that could compensate for the lack of base stacking. In addition to base-on-deoxyribose stacking and intra-cytidine C-H...O hydrogen bonds, this analysis reveals the presence of a hitherto unobserved, systematic intermolecular C-H...O hydrogen bonding network between the deoxyribose sugar moieties of antiparallel backbones in the four-stranded molecule.

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Mismatch repair in Xenopus egg extracts: DNA strand breaks act as signals rather than excision points.

In Xenopus egg extracts, DNA strand breaks (nicks) located 3' or 5' to a mismatch cause an overall 3-fold stimulation of the repair of the mismatch in circular heteroduplex DNA molecules. The increase in mismatch repair is almost entirely due to an increase in repair of the nicked strand, which is stimulated 5-fold. Repair synthesis is centered to the mismatch site, decreases symmetrically on both sides, and its position is not significantly altered by the presence of the nick. Therefore, it appears that in the Xenopus germ cells, the mismatch repair system utilizes nicks as signals for the induction and direction of mismatch repair, but not as the start or end point for excision and resynthesis.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.

Activating transcription from single stranded DNA.

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.

DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.

The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.