Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

Reichard, Peter; Eliasson, R.; Ingemarson, Rolf; Thelander, Lars

doi:10.1074/jbc.m005337200

Cited by 65 publications

(84 citation statements)

References 28 publications

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“…The allosteric activity site controls overall RNR activity: Binding of dATP inactivates the enzyme, whereas ATP stimulates catalysis (104). Substitution of a highly conserved amino acid (D57N) in the allosteric activity site of yeast and mammalian RNR leads to elimination of dATP feedback inhibition and an elevated nucleotide pool (38,104). These studies provide proof of concept that an ATP mimic with higher affinity for the activity site could boost dNTP pools during S-phase.…”

Section: Discussionmentioning

confidence: 58%

“…The allosteric specificity site controls NDP selection in the catalytic site, ensuring that dNTP pools remain balanced. The allosteric activity site controls overall RNR activity: Binding of dATP inactivates the enzyme, whereas ATP stimulates catalysis (104). Substitution of a highly conserved amino acid (D57N) in the allosteric activity site of yeast and mammalian RNR leads to elimination of dATP feedback inhibition and an elevated nucleotide pool (38,104).…”

Section: Discussionmentioning

confidence: 99%

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dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Williams

Marjavaara

Knowels

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) e mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol e depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol e proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol e base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol e mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.DNA replication and repair | polymerase fidelity | cancer | lethal mutagenesis

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Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Williams

Marjavaara

Knowels

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…This result is in contrast to a study of RNR from mouse with a mutation at the corresponding position (D57N). Here the mutation at the allosteric activity site led to a decreased binding of dATP also at the specificity site (25). However, in a recent study on the mouse enzyme with the D57N mutation, Kashlan and Cooperman (26) did not see any effect on binding to the specificity site.…”

Section: Discussionmentioning

confidence: 97%

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Birgander

Kasrayan

Sjöberg

2004

Journal of Biological Chemistry

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Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.A key enzyme of nucleic acid metabolism in the cell is ribonucleotide reductase. This enzyme, abbreviated RNR, 1 catalyzes the reaction where ribonucleotides are converted to deoxyribonucleotides, thus providing the cell with all components of DNA. RNR activity is essential for DNA synthesis and repair, but is also in need of a strict control system. An uncontrolled production of DNA precursors is devastating to the cell since the mutation frequency is increased at aberrant concentrations of dNTPs (1). In prokaryotic as well as most eukaryotic RNRs belonging to the class Ia enzymes, this is solved by an allosteric regulation controlling both the substrate specificity and the overall activity. Other classes of RNR, denoted Ib, II, and III, all perform the same reaction but operate at different conditions and via slightly different catalytic mechanisms (for a review, see Ref.2). Class Ib and II RNRs are allosterically regulated in a similar manner, and the class III enzymes are regulated approximately by the same effectors as class I RNRs (Fig. 1). In this study, all studies concern the class Ia RNR from Escherichia coli.The E. coli class Ia enzyme is composed of two dimeric proteins with different properties. The larger protein, denoted R1, contains the catalytic site and two types of allosteric sites. The catalytic mechanism involves advanced chemistry that is accomplished by a stable amino acid radical that originates from the smaller subunit, denoted R2. The fre...

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“…dTTP activated GDP reduction much more strongly than ADP reduction, whereas dGTP had the converse effect. The activation of ADP reduction by dTTP and the activation of GDP reduction by dGTP are effects not seen in single-substrate analysis of calf thymus RNR (10) or recombinant mouse RNR (11). dATP slightly activated ADP reduction and strongly inhibited CDP and UDP reduction, whereas GDP reduction was undetectable.…”

Section: Regulatory Effects Of Individual Nucleosidementioning

confidence: 97%

“…Because ADP in this experiment is expected to bind to the catalytic site as well as to possible other sites, we modified the protocol by asking whether ADP could inhibit dATP binding under conditions where little if any ADP was bound at the catalytic site. This was accomplished by running the binding assays in the presence of CDP at a constant concentration of 320 M, 10-fold higher than the reported K m for CDP with a mammalian RNR (11), so that ADP would be largely displaced from catalytic sites. Under these conditions also (Fig.…”

Section: Fig 4 Effects Upon Mouse Rndp Reductase Specificity Of Varmentioning

confidence: 99%

Mouse Ribonucleotide Reductase Control

Chimploy

Mathews

2001

Journal of Biological Chemistry

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Using ribonucleotide reductase encoded by vaccinia virus as a model for the mammalian enzyme, our laboratory developed an assay that allows simultaneous monitoring of the reduction of ADP, CDP, GDP, and UDP. That study found ADP reduction to be specifically inhibited by ADP itself. To learn whether this effect is significant for cellular regulation, we have analyzed recombinant mouse ribonucleotide reductase. We report that allosteric control properties originally described in single-substrate assays operate also under our four-substrate assay conditions. Three distinctions from the vaccinia enzyme were seen: 1) higher sensitivity to allosteric modifiers; 2) higher activity with UDP as substrate; and 3) significant inhibition by ADP of GDP reduction as well as that of ADP itself. Studies of the effects of ADP and other substrates upon binding of effectors indicate that binding of ribonucleoside diphosphates at the catalytic site influences dNTP binding at the specificity site. We also examined the activities of hybrid ribonucleotide reductases, composed of a mouse subunit combined with a vaccinia subunit. As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity. Surprisingly, a mouse R1/vaccinia R2 hybrid was more active than either mouse R1/R2 or vaccinia R1/R2, possibly explaining why mutations affecting vaccinia ribonucleotide reductase have only small effects upon viral DNA replication.Ribonucleotide reductase (RNR), 1 which catalyzes the first reaction committed to DNA synthesis, is distinctive in many ways. First, the interplay between two different allosteric sites evidently acts to ensure that the four deoxyribonucleotide products of the enzyme are synthesized at rates proportional to the base composition of the genome of the organism (1). Because deoxyribonucleotides have no known functions in eukaryotic cells except as DNA precursors, it makes good metabolic sense for rates of synthesis and utilization to be balanced.Second and less thoroughly investigated, the four ribonucleotide substrates are reduced at one active site. To what extent does competition among the four ribonucleotide substrates for binding to this site contribute toward the ability of the enzyme to act in concert with the need of the cell for dNTPs? It was partly for this reason that our laboratory developed an assay protocol that allows all four RNR reactions to be monitored simultaneously, in one reaction mixture (2, 3). Our results, obtained with ribonucleotide reductases encoded by bacteriophage T4 (2) and vaccinia virus (3) support the conclusion that ribonucleotide substrate concentrations are equal in importance to concentrations of nucleoside triphosphate effectors as determinants of the ability of the enzyme to produce its four products at rates proportional to the base composition of the genome.We have used the vaccinia virus enzyme as a model for mammalian ribonucleoside diphosphate reductases, because of its close structural relationship to the mammalian enzymes and because the viral enzyme sub...

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Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

Cited by 65 publications

References 28 publications

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Mouse Ribonucleotide Reductase Control

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