Luteus DNA Research Articles

The Binding Mechanisms of Antibodies to DNA from Healthy Subjects and Patients with Systemic Lupus Erythematosus: The Role of Monogamous Bivalency and Fc Dependence.

Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site; the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the activity of intact IgG with Fab and F(ab')2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf thymus or Micrococcus luteus These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab')2 preparations from both SLE patients and HS showed binding to M. luteus DNA; F(ab')2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA. Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to bacterial DNA does not require the Fc region.

Isolation and Rationale for the Formation of Isomeric Decarbamoylmitomycin C- N6-deoxyadenosine Adducts in DNA.

Mitomycin C (MC) is an anticancer agent that alkylates DNA to form monoadducts and interstrand cross-links. Decarbamoylmitomycin C (DMC) is an analogue of MC lacking the carbamate on C10. The major DNA adducts isolated from treatment of culture cells with MC and DMC are N2-deoxyguanosine (dG) adducts and adopt an opposite stereochemical configuration at the dG-mitosene bond. To elucidate the molecular mechanisms of DMC-DNA alkylation, we have reacted short oligonucleotides, calf thymus, and M. luteus DNA with DMC using biomimetic conditions. These experiments revealed that DMC is able to form two stereoisomeric deoxyadenosine (dA) adducts with DNA under bifuntional reduction conditions and at low temperature. The dA-DMC adducts formed were detected and quantified by HPLC analysis after enzymatic digestion of the alkylated DNA substrates. Results revealed the following rules for DMC dA alkylation: (i) DMC dA adducts are formed at a 48- to 4-fold lower frequency than dG adducts, (ii) the 5'-phosphodiester linkage of the dA adducts is resistant to snake venom diesterase, (iii) end-chain dA residues are more reactive than internal ones in duplex DNA, and (iv) nucleophilic addition by dA occurs on both faces of DMC and the ratio of stereoisomeric dA adducts formed is dependent on the end bases located at the 3' or 5' position. A key finding was to discover that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by DMC: at 0 °C, both dA and dG alkylation occur, whereas at 37 °C, DMC preferentially alkylates dG residues.

Binding affinities of folic acid and related pterins with biological macromolecules under physiological conditions

Abstract Folic acid and pterin derivatives are important heterocyclic compounds found in a variety of biological systems and have been shown to be photochemically active. Understanding the amount of binding that various pterins have with biological macromolecules under physiological conditions is important in predicting what specific biomolecules will bind with pterins and may, therefore, result in photochemical damage from charge-transfer reactions. The relative binding of folic acid, or pteroyl-L-glutamic acid (PteGlu), 6-methylpterin (Mep), 6-hydroxymethylpterin (Hmp), 6-formylpterin (Fop), and 6-carboxypterin (Cap) with bovine serum albumin (BSA), electrically neutral lipid (ENL), polyguanylic acid (Poly G), polycytidylic acid (Poly C), polyadenylic acid (Poly A), polythymidylic acid (Poly T), Micrococcus luteus DNA (72% GC), Escherichia coli DNA (50% GC), calf thymus DNA (42% GC), and Clostridium perfrigens DNA (27% GC) in neutral phosphate buffer were studied. Our results indicate that PteGlu demonstrated strong binding to neutral lipids, while the other pterins showed minimal binding, and BSA had a significant binding to PteGlu, Cap, and especially Fop. Our results also reveal a high affinity for DNA by PteGlu, which suggests that a relatively high percentage of folic acid is bound to DNA before photochemistry occurs.

The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.

Unusual salt-induced behaviour of guanine-rich natural DNA evidenced by dynamic light scattering

The appearance of the slow mode, revealed by dynamic light scattering (DLS) measurements in Micrococcus luteus DNA with high GC content, and the effect of guanine sequences on changes of DNA physical state and conformational transitions were investigated. We used two different spectroscopic approaches: DLS, to evidence the relatively slowly diffusing particles arising at high salt concentration, ascribable to the formation of large unspecific molecular aggregates, and circular dichroism spectroscopy, to identify these entities. Our results bring us to conclude that a peculiar, unconventional, structural transition, due to the presence of long guanine stretches, in a well-defined experimental condition, can occur. We comment on the biological implications to detect, by spectroscopic measurements, such an unusual structure involved in the stability, protection and replication maintenance along the human telomeric G-rich strand.

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)(2)], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)(2)] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100mA beam current of 0.5-μW power varied from 0.6h(-1) for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)(2)] and high salt poly[(dA-dT)(2)]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.

Effects of glutathione on alkylation and cross-linking of DNA by mitomycin C. Isolation of a ternary glutathione-mitomycin-DNA adduct.

Mitomycin C (MC), a clinically used antitumor antibiotic, is known to alkylate DNA monofunctionally, and to generate DNA interstrand cross-links by bifunctional alkylation. Both processes are dependent on the reductive activation of MC. Glutathione (GSH) was shown here to cause three types of changes in the pattern of alkylation of DNA by MC: (i) GSH caused a decrease of both the overall covalent binding ratio of MC to Micrococcus luteus DNA and the extent of interstrand cross-linking of 32P-pBR322 DNA, as the concentration of GSH was increased in the reaction media. Approximately 50% inhibition of cross-linking was observed at 20 mM GSH. It is likely that the inhibition is caused by the formation of MC-GSH conjugates competing with DNA alkylation, since both processes are triggered by reductive activation of MC [Sharma, M., and Tomasz, M. (1994) Chem. Res. Toxicol. (preceding paper in this issue)]. (ii) GSH causes a switch from monofunctional to bifunctional activation of MC by the prototype "monofunctional" MC-activating agents H2/PtO2 and NADPH:cytochrome c reductase/NADPH. This was seen by the predominance of bisadducts (i.e., cross-linked adducts) instead of the usual monoadducts in the enzymatic digests of MC-DNA complexes formed in the presence of GSH, as analyzed by HPLC. This finding suggests that GSH participates in the bifunctional activation of MC in vivo. (iii) A ternary MC-GSH-DNA adduct (6) was formed in the presence of GSH both with M. luteus DNA and with a synthetic duplex oligonucleotide; in this adduct the mitosene C1 is linked to N2 of guanine and the mitosene C10 is linked to GSH via sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific hypomethylation of c- myc protooncogene in liver nodules and inhibition of DNA methylation by N-nitrosomorpholine

The protooncogene c- myc was investigated in N-nitrosomorpholine-induced rat liver nodules to elucidate the role of altered DNA methylation in chemical carcinogenesis. Furthermore, Micrococcus luteus DNA and chicken erythrocyte DNA were modified in vitro by reactive metabolites of N-nitrosomorpholine, generated by P450-dependent monooxygenases. The modified DNAs were less methylated in vitro than control DNAs by DNA-(cytosine-5)-methyltransferase (DNA methylase). The DNA methylase assay and 32P-postlabeling analysis revealed lowered levels of DNA methylation in nodular DNA. In nodular tissue, c- myc messenger RNA levels were found to be increased compared to normal liver. DNA methylation analysis using the restriction endonucleases HpaII/MspI indicated hypomethylation in the first intron of c- myc DNA in liver nodules. The results suggest that genotoxic lesions may cause stably inherited, aberrant DNA methylation patterns which may be responsible for site-specific hypomethylation of the c- myc protooncogene in liver nodules.

Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogs

Conformation detection and base dynamics of spin-labeled Z-DNA have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The two synthesized and characterized probes used in this study were C(5)-nitroxide-labeled 2'-deoxycytidine 5'-triphosphates, pppDCAT and pppDCAVAT, which serve as suitable substrates for Micrococcus luteus DNA polymerase. Enzymatic incorporation of these probes into (dG-dC)n yields the EPR-active alternating copolymers (dG-dC,DCAT)n and (dG-dC,-DCAVAT)n. These polymers assume typical B- and Z-DNA conformations under respective low (0.1 M NaCl) and high (4.5 M NaCl) salt conditions, as evidenced by their UV-circular dichroism spectra. The EPR line shape of (dG-dC,DCAT)n in Z-form is unique and significantly different from the B-form EPR spectrum. A similar observation is made for (dG-dC,DCAVAT)n. Thus, the EPR line shapes of these spin-labeled DNAs are indicative of their local conformations. The EPR spectra, analyzed with a previously published motional model [Kao, S.-C., Polnaszek, C.F., Toppin, C.R., & Bobst, A.M. (1983) Biochemistry 22, 5563-5568], indicate tau perpendicular values of 4 and 7 ns for the B- and Z-forms, respectively. Therefore, the base dynamics of Z-DNA are about two times slower than in B-DNA.

In vitro inhibition of bacterial DNA gyrase by cinodine, a glycocinnamoylspermidine antibiotic

Cinodine, a broad-spectrum glycocinnamoylspermidine antibiotic, binds to DNA and irreversibly inhibits bacterial and phase DNA synthesis. Cinodine was found to inhibit the activity of Micrococcus luteus DNA gyrase in vitro, but it did not inhibit the activities of two other DNA-binding enzymes, namely, topoisomerase I and BamHI. Although we cannot yet conclude that DNA gyrase is an intracellular target of the drug, in vitro inhibition of the enzyme by cinodine appears to be specific.

Replication, Transcription and Nuclease Digestion of the Unusual X-DNA Double Helix of Poly(amino2dA-dT)

The alternating copolymer poly(amino2dA-dT) isomerizes into the unusual X-DNA double helix at low-salt aqueous conditions (Vorlicková et al., J. Biomolec. Struct. Dyn. 6, 503-510 (1988)). This observation allowed us to start studies on how the X-DNA is recognized, copied and hydrolyzed by various enzymes. In the present paper X-DNA replication, transcription and digestion by various polymerases and nucleases, respectively, are examined and compared to appropriate controls. It is found that X-DNA is a poor primer-template for DNA synthesis by the E. coli Klenow DNA polymerase (12% of the activity observed with B-DNA), the Micrococcus luteus DNA polymerase I (25%) and the AMV reverse transcriptase (51%). In contrast, X-DNA is a better template by 74% than B-DNA for calf thymus DNA polymerase alpha. For transcription by E. coli RNA polymerase enzyme poly(amino2dA-dT) did not serve as a template at all in either B or X conformation. Poly(amino2dA-dT) in its B form proved to be much more stable than poly(dA-dT) against hydrolysis by pancreatic DNase and snake venom phosphodiesterase. Formation of the X conformation in poly(amino2dA-dT) decreased this large difference in nuclease stability.

An electron paramagnetic resonance probe to detect local Z-DNA conformations

We present spectroscopic evidence for an electron paramagnetic resonance (EPR) probe to detect local Z-DNA conformations in synthetic DNA. A spin labeled deoxycytidine-5′-triphosphate (pppDCAT) was co-polymerized with Micrococcus luteus DNA polymerase to yield the spin active alternating co-polymer (dG-dC,DCAT)n. The EPR spectrum of (dG-dC,DCAT)n in the Z-DNA conformation indicates a decrease in the local base dynamics by about a factor of two as compared to that computed for B-DNA. A control experiment conducted with (dA-dT, DUAT)n under similar salt conditions rules out the possibilty of observing salt induced artifacts.

Micrococcus luteus, a bacterium with a high genomic G+C content, contains Escherichia coli-type promoters

The G + C content of Micrococcus luteus DNA is 74%, which is much higher than that of Escherichia coli (about 50%). In order to understand the influence of GC-directed mutation pressure (GC pressure) on the promoter structure, the initiation sites for both in vitro and in vivo transcription of M. luteus streptomycin (str) and spectinomycin (spc) operons were identified by the reverse transcriptase mapping method. The promoter sequences of M. luteus are similar to those of E. coli, but have significantly higher G + C contents. In an in vitro run-off transcription assay using truncated DNA templates, RNA polymerases from both M. luteus and E. coli were able to transcribe correctly from both the str promoter and the spc major promoter of M. luteus.

A possible role of chromatin and tightly-bound chromatin proteins on enzyme-catalyzed methylation of DNA

Upon extensive digestion with DNAaseI of placenta chromatin matrix, previously "stripped" from its loosely-bound components by high-salt extraction, a fraction is obtained that contains almost no endogenous DNA methylase activity but whose DNA, if still included in this whole fraction--not if it has been purified to a protein-free condition--is a good substrate for externally added enzyme. This chromatin matrix can even cause a significant stimulation of methylation of single-stranded Micrococcus luteus DNA by placental methylase. In vivo, this phenomenon may have possible counterparts in the existence of highly-methylated regions of chromatin loops that appear to be protected by tightly-bound protein components from digestion of the "stripped loops" with DNAaseI.

Incision‐Excision DNA Repair Model: A Theoretical Analysis of the Effect of Enzyme Concentrations on DNA Repair

AbstractRelationships between the concentration of exonuclease, DNA polymerase, and DNA ligase participating in the DNA incision‐excision repair pathway have been derived by applying steady state conditions to concentration of the species of interest. The analysis utilizes concentrations of damage excised, gapped DNA (DNAm—n) and gap filled unligated DNA (DNAm) at the steady state as the parameters to measure the extent of DNA repair. The concentrations of DNAm—n and DNAm have been expressed in terms of experimentally determinable constants such as Km, catalytic constants for the enzymes, enzyme, and substrate DNA concentrations, making it possible to verify the theoretical predictions. The analysis predicts a low ratio of exonuclease to polymerase to observe any significant repair activity, a conclusion consistent with in vitro observation of Hamilton et al. (1974) that significant DNA repair is observed when exonuclease: polymerase ratio is 0.5. The analyis predicts fairly large concentrations of DNA ligase to minimize DNAm concentration at the steady state. Under specific conditions, reversal of ligation has been predicted and has been suggested as a possible mechanism of initiation of multi‐centered DNA replication in eukaryotes. This suggestion has been substantiated by experimental demonstration that DNA nicked in the presence of DNA ligase and AMP is substrate for M. Luteus DNA polymerase but not for T4 DNA polymerase, suggesting that adenylated DNA intermediate is formed and only cleaved by 5′—3′ exonuclease which is present in vivo, and has been isolated and characterized. The relationships have been numerically evaluated for different enzyme concentrations using derived and literature values for the constants. The potential application of these results for in vitro introduction of “errors” into DNA has been indicated.

Micrococcus luteus homolog of theEscherichia coli uvrA gene: Identification of a mutation in the UV-sensitive mutant DB7

Restriction fragments of Micrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in an Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with the E. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of the M. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of the E. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinicapyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to the E. coli UvrABC excinuclease.

Mechanism of monofunctional and bifunctional alkylation of DNA by mitomycin C.

The relative amounts of monofunctional and bifunctional alkylation products of DNA with mitomycin C (MC) depend on whether one or both masked alkylating functions of MC are activated reductively; adduct 8 is the result of one function and adducts 7 and 9, formed as a pair, are the result of both functions being activated [Tomasz, M., Lipman, R., Chowdary, C., Pawlak, J., Verdine, G. L., & Nakanishi, K. (1987) Science (Washington, D.C.) 235, 1204-1208]. To determine the mechanism governing this differential reactivity of MC with DNA, MC-Micrococcus luteus DNA complexes formed under varying conditions in vitro were digested to nucleosides and adducts. Adduct distribution, analyzed by high-performance liquid chromatography, served as the measure of monofunctional and bifunctional activation. H2/PtO2 and xanthine oxidase/reduced nicotinamide adenine dinucleotide (NADH) activated MC mostly monofunctionally, and Na2S2O4 activated the drug bifunctionally under comparable conditions. Excess MC selectively suppressed, but excess PtO2 selectively promoted, bifunctional activation by H2/PtO2; excess xanthine oxidase and/or NADH also had promoting effects. O2 tested in the Na2S2O4 system was inhibitory. 10-Decarbamoyl-MC acted strictly monofunctionally under all conditions. Monoadducts bound to DNA were converted to bis adducts upon rereduction. A mechanism with the following features was derived: (i) Activation of MC at C-1 and C-10 is sequential (C-1 first). (ii) A one-time reduction is sufficient for both. (iii) Activation of the second function may be selectively inhibited by kinetic factors or O2. (iv) 7 and 9 are coproducts of bifunctional activation; their ratio depends on the DNA base sequence. (v) Activation of the second function involves an iminium intermediate. Direct applications to the action of MC in vivo are discussed.

Coumarins as inhibitors of bacterial DNA gyrase.

Chlorobiocic acid and 3-(carbobenzoxyamino)-4,7-dihydroxy-8-methylcoumarin were identified as two new inhibitors of Micrococcus luteus DNA gyrase. Both compounds possess weak antibacterial activity against whole M. luteus cells which indicates that they probably lack efficient transport functions to penetrate the cell envelope.

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S- adenosyl- l-methionine . The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.

Isolation and Structure of a Covalent Cross-Link Adduct Between Mitomycin C and DNA

A DNA cross-link adduct of the antitumor agent mitomycin C (MC) to DNA has been isolated and characterized; the results provide direct proof for bifunctional alkylation of DNA by MC. Exposure of MC to Micrococcus luteus DNA under reductive conditions and subsequent nuclease digestion yielded adducts formed between MC and deoxyguanosine residues. In addition to the two known monoadducts, a bisadduct was obtained. Reductive MC activation with Na2S2O4 (sodium dithionite) leads to exclusive bifunctional alkylation. The structure of the bisadduct was determined by spectroscopic methods that included proton magnetic resonance, differential Fourier transform infrared spectroscopy, and circular dichroism. Formation of the same bisadduct in vivo was demonstrated upon injection of rats with MC. Computer-generated models of the bisadduct that was incorporated into the center of the duplex B-DNA decamer d(CGTACGTACG)2 indicated that the bisadduct fit snugly into the minor groove with minimal distortion of DNA structure. A mechanistic analysis of the factors that govern monofunctional and bifunctional adduct formation is presented.