Protein–DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single‐stranded DNA‐binding protein

Kim, JuHyun; Wheeler, Linda J.; Shen, Rongkun; Mathews, Christopher K.

doi:10.1111/j.1365-2958.2004.04486.x

Cited by 18 publications

(20 citation statements)

References 30 publications

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“…This and other results led us to propose that NDP reductase has not just a functional but a structural implication in the replication process. Extending Norris' theoretical model [18], we call it the replication hyperstructure [12,13], resulting from the association of the replication factory and Mathews' dNTPsynthesizing complex [4,5]. The greatest difference in the effects caused by the chemical or thermal inactivation of NDP reductase was observed in the cell size and nucleoid structure.…”

Section: Discussionmentioning

confidence: 99%

“…This pool would permit replication for no longer than half a minute [2,3]. Channelling of the biosynthesis and compartmentation of the precursors have been proposed as explanations of how this shortage may be circumvented [4,5]. To satisfy the changing demand for the four deoxynucleotides, NDP reductase must be closely associated with the replication machinery.…”

Section: Introductionmentioning

confidence: 99%

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Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation

Riola

Guarino

Guzmán

et al. 2007

Cellular and Molecular Biology Letters

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NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the nonpermissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation

Riola

Guarino

Guzmán

et al. 2007

Cellular and Molecular Biology Letters

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show abstract

“…Glutathione S-Transferase Pulldown Assays-GST fusion proteins were prepared, and GST pulldown experiments carried out, as described in previous publications from this laboratory (11,20). Fusion proteins made for this study included gp1 (deoxyribonucleoside monophosphate kinase), gp42 (dCMP hydroxymethylase), gpfrd (dihydrofolate reductase), and E. coli nucleoside diphosphate kinase.…”

Section: Methodsmentioning

confidence: 99%

Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex

Kim

Shen

Olcott

et al. 2005

Journal of Biological Chemistry

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Adenylate kinase, which catalyzes the reversible ATPdependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720 -5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phagecoded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.Adenylate kinase catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP. The reaction is involved in the de novo biosynthesis of adenine nucleotides, and it is also thought to participate in adjusting adenine nucleotide levels to meet the energy demands of a cell (1). In addition, there is evidence that the enzyme in Escherichia coli participates in phospholipid biosynthesis, although the specific involvement has not been defined (2). Temperature-sensitive mutations in adk, the structural gene for adenylate kinase, cause defective phospholipid synthesis when bacteria are grown at a nonpermissive temperature (3).A novel capability for E. coli adenylate kinase was described when Lu and Inouye (4) found that the wild-type adk gene could complement a site-specific disruption of ndk, the structural gene for nucleoside diphosphate kinase. Lu and Inouye (5) showed that adenylate kinase could catalyze the conversion of nucleoside diphosphates to triphosphates. Experiments in our laboratory (5) showed that the phosphate donor for at least some of these reactions was ADP. So, instead of catalyzing the well known reaction, 2ADP º AMP ϩ ATP, the enzyme was evidently substituting a different nucleoside diphosphate for one of the two ADPs:Both NDP 1 kinase and adenylate kinase were shown, some ye...

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“…Recently, new approaches supporting the associations between dNTP synthesis enzymes, DNA, and the replication complex in T4-infected cells (14) and in E. coli (10,24) have been described. The data provided by the present work would be consistent with the presence of the NDP reductase at the replication fork in vivo and represent independent support for the presence of NDP reductase as a structural and functional component of the replication hyperstructure (10), as they show the occurrence of replication fork arrest in the presence of NDP reductase encoded by nrdA101 allele.…”

Section: Discussionmentioning

confidence: 99%

Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia coli

2007

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The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec ؉ context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA ؉ strain growing under limited (2-g/ml) or under optimal (5-g/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.Ribonucleoside diphosphate reductase (NDP reductase) is the only specific enzyme required for the enzymatic formation of deoxyribonucleotides (dNTPs), the precursors of DNA synthesis in Escherichia coli. NDP reductase is a 1:1 complex of two nonidentical subunits called proteins R1 and R2, encoded by genes nrdA and nrdB, respectively (for a review, see reference 3). The best-known defective NDP reductase mutant of E. coli contains a thermolabile R1 subunit encoded by the nrdA101 allele. The activity of the enzyme measured in crude extracts of nrdA101 strains is limited to 6% of the wild-type activity at 25°C (6), and the dNTP pool is lower than wild type even at permissive temperatures (16). Our laboratory has shown that the presence of the nrdA101 allele lowers the replication rate of the mutant strain at the permissive temperature, as a nrdA101 mutant replicates the chromosome in 154 min at 30°C, while a nrdA ϩ strain does so in 98 min (10). Regarding the detrimental effect of the nrdA101 allele on the activity of the enzyme, this DNA replication effect is assumed to be due to the decrease in the NDP reductase activity as a dNTP provider. However, NDP reductase has been proposed as a component of the replication hyperstructure (10), and consequently the structure of the NDP reductase encoded by the nrdA101 allele might provoke a structural alteration of the replication hyperstructure that could also contribute to the lengthening of the C period in the mutant. A possible consequence of an altered replication hyperstr...

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Protein–DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single‐stranded DNA‐binding protein

Cited by 18 publications

References 30 publications

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation

Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex

Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia coli

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