Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.
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The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties. (+info)
Identifying the bicyclomycin binding domain through biochemical analysis of antibiotic-resistant rho proteins.
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Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket. (+info)
The formation of DNA interstrand cross-links by a novel bis-[Pt2Cl4(diminazene aceturate)2]Cl4.4H2O complex inhibits the B to Z transition.
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We present data demonstrating that the cytotoxic compound [Pt2Cl4(diminazene aceturate)2]Cl4.4H2O (Pt-berenil) circumvents cisplatin resistance in ovarian carcinoma cells. The analysis of the interaction of Pt-berenil with linear and supercoiled DNA indicates that this compound induces the formation of a large number of covalent interstrand cross-links on DNA and that this number is significantly higher than that produced by cis-diamminedichloroplatinum(II) (cis-DDP). Renaturation experiments, interstrand cross-link assays, and electron microscopy indicate that the kinetics of DNA interstrand cross-link formation caused by Pt-berenil binding is faster than that caused by cis-DDP at similar levels of platinum bound to DNA. Furthermore, the number of DNA interstrand cross-links in Pt-berenil-DNA complexes is influenced by supercoiling. Circular dichroism experiments show that Pt-berenil strongly inhibits the B-DNA-to-Z-DNA transition of poly(dG-m5 dC). poly(dG-m5dC) at salt concentrations (3 mM MgCl2) at which the native methylated polynucleotide readily adopts the Z-DNA conformation, which suggests that the induction of interstrand cross-links by Pt-berenil inhibits the Z-DNA transition. On the basis of these results, we propose that bis(platinum) compounds with structure similar to Pt-berenil may act as blockers of DNA conformational changes and may also display activity in cisplatin-resistant cells. (+info)
Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.
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Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells. (+info)
Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity.
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With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism. (+info)
The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.
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Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method. (+info)
Phase variation in Helicobacter pylori lipopolysaccharide due to changes in the lengths of poly(C) tracts in alpha3-fucosyltransferase genes.
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The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that the two alpha3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 alpha3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the alpha3-fucosylation precedes alpha2-fucosylation [corrected], an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host. (+info)
The mechanism of ATP hydrolysis at the noncatalytic sites of the transcription termination factor Rho.
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Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate. The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites. A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites. Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s(-1) at 18 degrees C. The apparent bimolecular rate constant for ATP binding was determined as 5.4 x 10(5) M(-1) s(-1) in the presence of poly(C) RNA. The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s(-1). The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites. These results from studies of the rho protein are likely to be general to hexameric helicases. We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding. (+info)