scholarly journals The Escherichia coli dnaN159 Mutant Displays Altered DNA Polymerase Usage and Chronic SOS Induction

2004 ◽  
Vol 186 (20) ◽  
pp. 6738-6748 ◽  
Author(s):  
Mark D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Uv Light ◽  
Polymerase Activity ◽  
Sos Induction ◽  
Pol I ◽  
Absolute Dependence ◽  
Pol Iii ◽  
Partner Proteins

ABSTRACT The Escherichia coli β sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the β clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant β clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant β clamp protein (β159) is impaired for interactions with the α catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the β clamp might help to coordinate protein traffic at the replication fork.

scholarly journals Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated

2002 ◽  
Vol 184 (10) ◽  
pp. 2674-2681 ◽  
Author(s):  
Angela Borden ◽  
Paul I. O'Grady ◽  
Dominique Vandewiele ◽  
Antonio R. Fernández de Henestrosa ◽  
Christopher W. Lawrence ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Wild Type ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Pol V ◽  
Pol I ◽  
Cyclobutane Dimer ◽  
Dna Polymerase V ◽  
Pol Iii

ABSTRACT Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

scholarly journals Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

2007 ◽  
Vol 189 (13) ◽  
pp. 4688-4695 ◽  
Author(s):  
Robert W. Maul ◽  
Laurie H. Sanders ◽  
James B. Lim ◽  
Rosemary Benitez ◽  
Mark D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Sliding Clamp ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I

ABSTRACT The Escherichia coli dnaN159 allele encodes a mutant form of the β-sliding clamp (β159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the β clamp stimulates the processivity of Pol I in vitro and that β159 is impaired for this activity. The reduced ability of β159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the β clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ΔpolA strain.

scholarly journals Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the β clamp

2002 ◽  
Vol 44 (5) ◽  
pp. 1331-1339 ◽  
Author(s):  
Gianfranco Grompone ◽  
Marie Seigneur ◽  
S. Dusko Ehrlich ◽  
Bénédicte Michel
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Polymerase Iii ◽  
Polymerase Iii

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

scholarly journals Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells.

2005 ◽  
Vol 52 (1) ◽  
pp. 139-147
Author(s):  
Michał Wrzesiński ◽  
Anetta Nowosielska ◽  
Jadwiga Nieminuszczy ◽  
Elzbieta Grzesiuk
Keyword(s):  
Escherichia Coli ◽  
Uv Light ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Uv Mutagenesis ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Induced Mutagenesis ◽  
Pol Iii

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

scholarly journals DNA Polymerase I Is Essential for Growth ofMethylobacterium dichloromethanicum DM4 with Dichloromethane

2000 ◽  
Vol 182 (19) ◽  
pp. 5433-5439 ◽  
Author(s):  
Martin F. Kayser ◽  
Michael T. Stumpp ◽  
Stéphane Vuilleumier
Keyword(s):  
Dna Repair ◽  
Dna Polymerase ◽  
Uv Light ◽  
Polymerase Activity ◽  
Dna Polymerase I ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Dichloromethane Dehalogenase ◽  
Polymerase I ◽  
Methylobacterium Dichloromethanicum

ABSTRACT Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase–glutathioneS-transferase. A mutant of the dichloromethane-degrading strain M. dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the polA mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into thepolA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The polA′-lacZtranscriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of M. dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.

scholarly journals The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

2016 ◽  
Vol 36 (6) ◽  
Author(s):  
Lorenzo Randi ◽  
Alessandro Perrone ◽  
Mirko Maturi ◽  
Fabrizio Dal Piaz ◽  
Michela Camerani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Deinococcus Radiodurans ◽  
Catalytic Properties ◽  
Polymerase Activity

We report in the present study on the catalytic properties of Deinococcus radiodurans DnaE polymerase, whose DNA elongation efficiency was compared with the homologous Escherichia coli polymerase. Contrary to the latter, the deinococcal enzyme was found to be strictly dependent on RecA recombinase.

Sign in / Sign up

Export Citation Format

Share Document