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1.
Genes Genet Syst ; 87(4): 221-31, 2012.
Article in English | MEDLINE | ID: mdl-23229309

ABSTRACT

Escherichia coli dinB encodes the translesion DNA polymerase DinB, which can inhibit progression of replication forks in a dose-dependent manner, independent of exogenous DNA damage. We reported previously that overproduction of DinB from a multicopy dinB plasmid immediately abolished ongoing replication fork progression, and the cells rapidly and drastically lost colony-forming ability, although the mechanisms underlying this lethality by severe replication fork stress remained unclear. Here, we show that the reduced colony-forming ability in the dinB-overexpressing cells is independent of the specific toxin genes that trigger programmed bacterial cell death when replication is blocked by depletion of the dNTP pool. After DinB abolished replication fork progression and colony-forming ability, most of the cells were still viable, as judged by fluorescent dye staining, but contained irregularly shaped nucleoids in which chromosomal DNA was preferentially lost in the replication terminus region relative to the replication origin region. Flow cytometric analysis of the cells revealed chromosomal damage and the eventual appearance of cell populations with less than single-chromosome DNA content, reminiscent of sub-G1 cells with lethal DNA content produced during eukaryotic apoptosis. This reduced DNA content was not observed after replication fork progression was quickly stopped in temperature-sensitive dnaB helicase mutant cells at a non-permissive temperature. Thus, the quick replication stop provoked by excess DinB uniquely generates temporarily viable but non-reproductive cells possessing a fatally depleted chromosomal content, which may represent one of the possible fates of an E. coli cell whose replication is overwhelmingly compromised.


Subject(s)
Cell Proliferation , Chromosome Aberrations , DNA Replication , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Cell Death , DnaB Helicases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Nucleotides/metabolism , Replication Origin , Up-Regulation
2.
Mol Microbiol ; 70(3): 608-22, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18761688

ABSTRACT

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.


Subject(s)
DNA Polymerase beta/metabolism , DNA Replication , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Chromosomes, Bacterial/genetics , Colony Count, Microbial , DNA Polymerase beta/genetics , DNA, Bacterial/biosynthesis , DNA, Bacterial/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Mutation , Oligonucleotide Array Sequence Analysis , Phenotype , Plasmids
4.
J Pharm Sci ; 92(1): 67-76, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12486683

ABSTRACT

We investigated the mechanism of hepatobiliary transport of a novel thromboxane A(2) receptor antagonist, [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335), and its taurine conjugate (Z-335-Tau) in normal Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs). The biliary excretion rate/unbound concentration in the cytosol (nu(bile)/C(u,cyt)) of Z-335 was markedly decreased in EHBRs, whereas nu(bile)/C(u,cyt) values for Z-335-Tau did not differ significantly between EHBRs and SDRs. These results suggest that biliary excretion of Z-335 involves mrp2, whereas Z-335-Tau is excreted by other transporters. The effects of inhibitors on the biliary excretion of Z-335 and Z-335-Tau were also examined in SDRs. After infusion of bromosulfophthalein (BSP), the nu(bile)/C(u,cyt) of Z-335 was significantly decreased, whereas that of Z-335-Tau decreased to 50% of control values by infusion of indocyanine green (ICG) or taurocholate. However, biliary excretion of Z-335-Tau was maintained at a highly concentrative. In conclusion, the biliary excretion of Z-335 involves mrp2, whereas Z-335-Tau is excreted into the bile by active transport systems that remain intact in EHBRs. The mdr2 and/or BSEP/spgp might contribute to a part of total biliary excretion of Z-335-Tau, however, these transporters have not played a major role in the biliary excretion of Z-335-Tau.


Subject(s)
Biliary Tract/metabolism , Indans/pharmacokinetics , Liver/metabolism , Receptors, Thromboxane/antagonists & inhibitors , Taurine/pharmacokinetics , Animals , Bile/metabolism , Biological Transport/physiology , Indans/chemistry , Male , Rats , Rats, Sprague-Dawley , Receptors, Thromboxane/metabolism , Taurine/chemistry , Xenobiotics/chemistry , Xenobiotics/pharmacokinetics
5.
Drug Metab Dispos ; 30(5): 498-504, 2002 May.
Article in English | MEDLINE | ID: mdl-11950778

ABSTRACT

To elucidate the transport system by which [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335) is taken up into the liver, we investigated the uptake characteristics of Z-335 in isolated rat hepatocytes. In addition, we estimated the hepatic uptake of Z-335 in intact rats under steady-state conditions and compared it with the in vitro uptake clearance. Uptake of Z-335 is highly concentrative (cell-to-medium concentration ratios were 21.2 at 0.5 min and 71.7 at 5 min), temperature-dependent, and sensitive to metabolic inhibitors, indicating that uptake is mediated by energy-dependent uphill transport. In the presence of metabolic inhibitors [carbonyl cyanide p-trifluoromethoxyphenylhydrazone and rotenone], uptake remained at 37 and 49% of the control value, respectively, suggesting that ATP-independent uptake contributes to the total uptake of Z-335. The concentration dependence of the initial uptake velocity indicated a two-component process, one saturable component, with a K(m) value of 45.6 microM and a V(max) value of 4.1 nmol/min/mg of protein, and a nonspecific diffusion clearance, with a P(dif) value of 8.3 microl/min/mg of protein. The contribution of the carrier-mediated uptake to the total uptake in a linear range was estimated as 91%. The in vivo hepatic intrinsic clearance (CL(int, app)) was comparable with that in vitro uptake clearance (PS(influx)) and indicated that the CL(int, app) of Z-335 at steady state is rate-limited by the uptake process. In conclusion, hepatic intrinsic clearance of Z-335 at steady state is rate-limited by the uptake process since Z-335 is efficiently taken up by an active transport mechanism, followed by metabolism or biliary excretion.


Subject(s)
Hepatocytes/drug effects , Indans/metabolism , Receptors, Thromboxane/antagonists & inhibitors , Animals , Anions/pharmacology , Bile Acids and Salts/pharmacology , Biological Transport, Active , Cations/pharmacology , Hepatocytes/metabolism , In Vitro Techniques , Indans/pharmacokinetics , Male , Rats , Rats, Sprague-Dawley , Sodium/metabolism
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