Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Abstract

Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea. Hydroxyurea (HU)1 inhibits DNA replication in cells that use the class I form of ribonucleotide reductase (RNR) by inactivating the tyrosyl radical required for enzyme activity (1). This inhibition is also observed in viruses such as vaccinia, which encodes a class I RNR that is closely related in structure and regulation to the mammalian cell RNRs. HU inhibits the de novo synthesis of deoxyribonucleotides, thereby starving the host cell and viral replication complexes for precursors. One might expect the inhibition of RNR by a drug like HU to result in an equivalent decrease in the rates of formation of all four products of the enzyme. However, when our laboratory measured the triphosphate forms of these products, the 2Ј-deoxyribonucleoside 5Ј-triphosphates (dNTPs) in vaccinia virus-infected cells, we found that hydroxyurea treatment had different effects upon the four dNTP pools; dATP was the most severely depleted, dCTP and dGTP were depleted to intermediate levels, and dTTP actually accumulated to some 2-fold over control values (2). Similar results had earlier been noted with uninfected mammalian cells (3-5). In vaccinia virus-infected cells, addition of deoxyadenosine to the culture medium, along with an adenosine deaminase inhibitor, reversed the inhibition of virus growth by HU (2). Also, labeling studies that estimated RNR flux rates in vivo confirmed that ADP reduction was the most severely affected of the four RNR activities. These observations suggested that the reduction of ADP is a specific biological target of hydroxyurea in vivo. This finding, in turn, has spurred attempts to ...

Allosteric Regulation of Vaccinia Virus Ribonucleotide Reductase, Analyzed by Simultaneous Monitoring of Its Four Activities

“…Possibly this could represent a mechanism to prevent the accumulation under certain circumstances of dATP, which has been shown at low concentrations in vitro to stimulate activations of proteases in a cascade of events leading to apoptosis (24) and which is also a potent inhibitor of DNA replication through its effect on ribonucleotide reductase. Inhibition by ADP could also help explain the phenomenon explored in the companion paper, namely, the specific inhibition of ADP reduction in vivo by hydroxyurea (11). If hydroxyurea inhibition of RNR causes all four rNDPs to accumulate in the short term, then the accumulation of ADP, the most abundant rNDP, could lead to more specific inhibition of ADP reductase in the longer term, with a consequent specific depletion of dATP pools.…”

“…The purification procedure for the vaccinia R2 subunit was essentially as reported (5), except for the incorporation of an additional radical reactivation step, described in the accompanying paper (11). Mouse R2 protein was purified by the same procedure used to purify the vaccinia R2 (5).…”

“…Mouse R2 protein was purified by the same procedure used to purify the vaccinia R2 (5). Purification of the vaccinia R1 protein was performed also as described in the accompanying paper (11).…”

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity

“…Hydroxyurea has been reported to destroy the tyrosyl free radical at the active site of the M2 protein subunit of ribonucleotide reductase, inactivating the enzyme (Yarbro, 1992). This blocks the de novo synthesis of deoxyribonucleotides (Hendricks and Matthews, 1998), synchronizing DNA synthesis in S-phase. If no excision repair occurs in Sphase (Bell et al, 1999), HD will cause greater damage to cells synchronized in S-phase because potentially no DNA repair may occur after the HD exposure until the cells have completed the cell cycle.…”

Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

“…Hydroxyurea (HU) is a compound that inhibits ribonucleoside dephosphate reductase and thereby blocks DNA synthesis (Hendricks and Mathews, 1998). This "classic" feature of HU has been widely used to induce intra-S and S/M checkpoints that delay progression through S phase and prevent mitotic entry due to the presence of incompletely replicated DNA.…”