The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper.

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, but (Phen)2Cu(I) predominates when ascorbate is used as the reducing agent. Substitution of ascorbate for thiol significantly enhances the rate of DNA cleavage by 1,10-phenanthroline + copper, without altering the sequence selectivity. We show that (Phen)2Cu(I) is the complex responsible for DNA cleavage, regardless of reducing agent, and that 1,10-phenanthroline and MPA compete for copper coordination sites. DNA cleavage in the presence of ascorbate also occurs under conditions where the mono-(1,10-phenanthroline)Cu(I) complex predominates (1:1 phenanthroline:copper ratio), but preferential cleavage was observed at a CCGG sequence and not at TAT sequences. The second phenanthroline ring of the (Phen)2Cu(I) complex appears essential for determining the T-3',5'-A sequence preferences of phenanthroline + copper when phenanthroline is in excess.     

Polyamide recognition-mass spectrometry for distinguishing hairpin DNA from coil DNA

The discrimination between hairpin DNA and coil DNA has been well achieved through polyamides as probes by electrospray ionization (ESI) mass spectrometry. ESI mass spectra showed that polyamides bind to hairpin DNA with high selectivity, and almost no binding with coil DNA. In addition, the noncovalent interaction between polyamides and hairpin DNA was also studied; the results show that hairpin DNA with longer stem and polyamides with more heterocycles have higher binding affinity and stability in gas phase.     

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes — a Ca²⁺/Mg²⁺-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca²⁺/Mg²⁺-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca²⁺/Mg²⁺-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg²⁺-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca²⁺/Mg²⁺-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.     

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.     

M<Superscript>3</Superscript>G: Maximum Margin Microarray Gridding

Background  

Complementary DNA (cDNA) microarrays are a well established technology for studying gene expression. A microarray image is obtained by laser scanning a hybridized cDNA microarray, which consists of thousands of spots representing chains of cDNA sequences, arranged in a two-dimensional array. The separation of the spots into distinct cells is widely known as microarray image gridding.     

Alpha-diaminobutyric acid-linked hairpin polyamides

A hairpin polyamide-chlorambucil conjugate linked by alpha-diaminobutyric acid (alpha-DABA) has been shown to have interesting biological properties in cellular and small animal models. Remarkably, this new class of hairpin polyamides has not been previously characterized with regard to energetics and sequence specificity. Herein we present a series of pyrrole-imidazole hairpin polyamides linked by alpha-DABA and compare them to polyamides containing the standard gamma-DABA turn unit. The alpha-DABA hairpins have overall decreased binding affinities. However, alpha-DABA polyamide-chlorambucil conjugates are sequence-specific DNA alkylators with increased specificities. Affinity cleavage studies of alpha-DABA polyamide-EDTA conjugates confirmed their preference for binding DNA in a forward hairpin conformation. In contrast, an unsubstituted glycine-linked polyamide prefers to bind in an extended binding mode. Thus, substitution on the turn unit locks the alpha-DABA polyamide into the forward hairpin binding motif.     

Coronavirus 3CL<Superscript><Emphasis Type="Italic">pro</Emphasis></Superscript>proteinase cleavage sites: Possible relevance to SARS virus pathology

Background  

Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods.     

Regulatory role of E-NTPase/E-NTPDase in Ca<Superscript>2+</Superscript>/Mg<Superscript>2+</Superscript> transport via gated channel

Background  

E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca²⁺ or Mg²⁺ with a pH optimum of 7.5 for the hydrolysis of extracellular NTP and NDP. It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered.     

Solid-phase synthesis of DNA binding polyamides on oxime resin

Control of the energetics and specificity of DNA binding polyamides is necessary for inhibition of protein-DNA complex formation and gene regulation studies. Typically, solid-phase methods using Boc monomers for synthesis have depended on Boc-beta-Ala-PAM resin which affords a beta-alanine-Dp tail at the C-terminus, after cleavage with N,N-dimethylaminopropylamine (Dp). To address the energetic consequences of this tail for DNA minor groove binding, we describe an alternative solid phase method employing the Kaiser oxime resin which allows the synthesis of polyamides with incrementally shortened C-terminal tails. Polyamides without Dp and having methyl amide tails rather than beta-alanine show similar affinity relative to the standard beta-Dp tail. The truncated tail diminishes the A,T base pair energetic preference of the beta-Dp tail which will allow a greater variety of DNA sequences to be targeted by hairpin polyamides.     

Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py–Im polyamides 1–5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k_a) of the Py–Im polyamides with their target DNA decreased with the number of Im in the Py–Im polyamides. The structures of four-ring Py–Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py–Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py–Im polyamide induced by the incorporation of Im reduces the association rate of Py–Im polyamides. This fundamental knowledge of the binding of Py–Im polyamides to DNA will facilitate the design of hairpin Py–Im polyamides as synthetic DNA-binding modules.     

Human <Emphasis Type="Italic">CCS</Emphasis> gene: genomic organization and exclusion as a candidate for amyotrophic lateral sclerosis (ALS)

Background  

Amyotrophic lateral sclerosis (ALS) is a progressive lethal disorder of large motor neurons of the spinal cord and brain. In approximately 20% of the familial and 2% of sporadic cases the disease is due to a defect in the gene encoding the cytosolic antioxidant enzyme Cu, Zn-superoxide dismutase (SOD1). The underlying molecular defect is known only in a very small portion of the remaining cases and therefore involvement of other genes is likely. As SOD1 receives copper, essential for its normal function, by the copper chaperone, CCS (Copper Chaperone for SOD), we considered CCS as a potential candidate gene for ALS.     

Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates

Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.     

DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators.

With the goal of developing artificial nucleases for DNA hydrolysis, metal-coordinating peptides have been tethered to a DNA-intercalating rhodium complex to deliver metal ions to the sugar-phosphate backbone. The intercalator, [Rh(phi)(2)bpy']Cl(3) [phi = 9,10-phenanthrenequinone diimine; bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine], provides DNA binding affinity, and a metal-binding peptide contributes reactivity. This strategy for DNA hydrolysis is a general one, and zinc(II)-promoted cleavage has been demonstrated for two widely different tethered metallopeptides. An intercalator coupled with a de novo-designed alpha helix containing two histidine residues has been demonstrated to cleave both supercoiled plasmid and linear DNA substrates. Mutation of this peptide confirms that the two histidine residues are essential for Zn(2+) binding and cleavage. Zinc(II)-promoted cleavage of supercoiled plasmid has also been demonstrated with an intercalator-peptide conjugate containing acidic residues and modeled after the active site of the BamHI endonuclease. Other redox-active metals, such as copper, have been delivered to DNA with our intercalator-peptide conjugates to effect oxidative chemistry. Copper cleavage experiments and photocleavage experiments with [Rh(phi)(2)bpy'](3+) complement the hydrolysis studies and provide structural information about the interactions between the tethered metallopeptides and DNA. Variation of the rhodium intercalator was also explored, but with a mismatch-specific intercalator, no site-specific hydrolysis was found. These experiments, in which the peptide, the metal cation, and the intercalator components of the conjugate are each varied, illustrate some of the issues involved in creating an artificial nuclease with DNA intercalators and metallopeptides.     

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Background  

V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca<Superscript>2+</Superscript> sensitivity but not on Ca<Superscript>2+</Superscript> sparks or BK channels

Background

In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels leading to lowered SMC [Ca²⁺]_i and vasodilation. Here we investigated whether Ca²⁺ sparks regulate SMC global [Ca²⁺]_i and diameter in the spiral modiolar artery (SMA) by activating BK channels.

Methods

SMAs were isolated from adult female gerbils, loaded with the Ca²⁺-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca²⁺ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca²⁺ signals and vascular diameter were analyzed.

Results

Ca²⁺ sparks and waves were observed in pressurized SMAs. Inhibition of Ca²⁺ sparks with ryanodine increased global Ca²⁺ and constricted SMA at 40 cmH₂O but inhibition of Ca²⁺ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH₂O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH₂O was abolished at 60 cmH₂O, consistent with a greater Ca²⁺-sensitivity of constriction at 40 cmH₂O than at 60 cmH₂O. When the Ca²⁺-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH₂O.

Conclusions

The results suggest that Ca²⁺ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca²⁺-sensitivity of constriction.