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Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase

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ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.


The Depletion of the Regulatory Subunit of Primase Is Specific to HUTreatment(A–C) The upper panel shows plating of 10-fold serial dilutions eithermock-treated (−) or treated (+) with (A) 200 J/m2 of UV light(254 nm) or (B) 30 μM or (C) 100 μM hydrogen peroxide for theindicated times. The lower panels contain western blots to determine levels ofprimase and TBP proteins.(D) Treatment of 13.5 μM recombinant primase (upper panel), MCM (middlepanel), or PolB1 (bottom panel) with the indicated concentrations of HU for 4 hrat 78°C leads to selective precipitation of primase. Following treatment,samples were centrifuged to separate soluble (s) and precipitated (p) materialand corresponding fractions analyzed by SDS-PAGE and stained with Coomassiebrilliant blue.
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Figure 6: The Depletion of the Regulatory Subunit of Primase Is Specific to HUTreatment(A–C) The upper panel shows plating of 10-fold serial dilutions eithermock-treated (−) or treated (+) with (A) 200 J/m2 of UV light(254 nm) or (B) 30 μM or (C) 100 μM hydrogen peroxide for theindicated times. The lower panels contain western blots to determine levels ofprimase and TBP proteins.(D) Treatment of 13.5 μM recombinant primase (upper panel), MCM (middlepanel), or PolB1 (bottom panel) with the indicated concentrations of HU for 4 hrat 78°C leads to selective precipitation of primase. Following treatment,samples were centrifuged to separate soluble (s) and precipitated (p) materialand corresponding fractions analyzed by SDS-PAGE and stained with Coomassiebrilliant blue.

Mentions: The data above indicate that initiation of DNA replication at all threeorigins is impacted by HU treatment, even though levels and chromatinassociation of WhiP, the initiator for oriC3, are unaffected byadministration of HU. Our observation that the regulatory subunit of primase,PriL, is selectively lost upon HU treatment could account for the loss of originfiring and cessation of fork progression that we observe. However, it isformally possible that the effects we see on PriL levels could be a consequenceof cell death, rather than a direct effect of HU treatment. To address thisconcern, we first subjected cells to two other genotoxic insults, UV treatmentand treatment with hydrogen peroxide. We exposed cells to 200 J/m2UV, and then grew them for 4 or 7 hr before plating serial dilutions to test forviability (Figure 6A). To preventphoto-reactivation, the cells were grown in the dark prior to plating (Götz et al., 2007; Fröls et al., 2007). Viability wasessentially unaffected by 4-hr growth in the dark after UV irradiation; however,growth for 7 hr prior to plating resulted in survival dropping by two orders ofmagnitude. Despite the differences in viability, western blotting revealed nosignificant changes in the absolute or relative levels of primase catalytic andregulatory subunits (Figure 6A). Similarly,treatment of cells with 30 or 100 μM hydrogen peroxide for 4 or 7 hrimpacted on viability (Figures 6B and 6C),with severity of impact on cell viability scaling with concentration andexposure time. This ranged from an approximately 10-fold reduction in the numberof viable cells with a 4-hr treatment with 30 μM hydrogen peroxide tocomplete loss of viability with administration of 100 μM hydrogenperoxide. Regardless of the impact on viability, relative levels of primasesubunits were unaltered by H2O2 (Figures 6B and 6C). Thus, the effects of HU on levels ofPriL appear to be specific to treatment with this agent, not simply aconsequence of cell death. To determine whether HU could directly impact primasestability, we tested the effect of hydroxyurea on the purified primase complexin vitro. As can be seen in Figure 6D, toppanel, incubation of the heterotrimeric PriSLX complex with 5 or 10 mM HU for 4hr leads to enhanced precipitation of the complex. Compared to the water-treatedcontrol, we observe a 16- or 20-fold enrichment of primase in the insolublematerial for 5 and 10 mM HU, respectively. Incubation of recombinant MCMhelicase or DNA polymerase PolB1 with HU had no discernable impact on thesolubility of these proteins (Figure 6D,lower panels).


Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase
The Depletion of the Regulatory Subunit of Primase Is Specific to HUTreatment(A–C) The upper panel shows plating of 10-fold serial dilutions eithermock-treated (−) or treated (+) with (A) 200 J/m2 of UV light(254 nm) or (B) 30 μM or (C) 100 μM hydrogen peroxide for theindicated times. The lower panels contain western blots to determine levels ofprimase and TBP proteins.(D) Treatment of 13.5 μM recombinant primase (upper panel), MCM (middlepanel), or PolB1 (bottom panel) with the indicated concentrations of HU for 4 hrat 78°C leads to selective precipitation of primase. Following treatment,samples were centrifuged to separate soluble (s) and precipitated (p) materialand corresponding fractions analyzed by SDS-PAGE and stained with Coomassiebrilliant blue.
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Related In: Results  -  Collection

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Figure 6: The Depletion of the Regulatory Subunit of Primase Is Specific to HUTreatment(A–C) The upper panel shows plating of 10-fold serial dilutions eithermock-treated (−) or treated (+) with (A) 200 J/m2 of UV light(254 nm) or (B) 30 μM or (C) 100 μM hydrogen peroxide for theindicated times. The lower panels contain western blots to determine levels ofprimase and TBP proteins.(D) Treatment of 13.5 μM recombinant primase (upper panel), MCM (middlepanel), or PolB1 (bottom panel) with the indicated concentrations of HU for 4 hrat 78°C leads to selective precipitation of primase. Following treatment,samples were centrifuged to separate soluble (s) and precipitated (p) materialand corresponding fractions analyzed by SDS-PAGE and stained with Coomassiebrilliant blue.
Mentions: The data above indicate that initiation of DNA replication at all threeorigins is impacted by HU treatment, even though levels and chromatinassociation of WhiP, the initiator for oriC3, are unaffected byadministration of HU. Our observation that the regulatory subunit of primase,PriL, is selectively lost upon HU treatment could account for the loss of originfiring and cessation of fork progression that we observe. However, it isformally possible that the effects we see on PriL levels could be a consequenceof cell death, rather than a direct effect of HU treatment. To address thisconcern, we first subjected cells to two other genotoxic insults, UV treatmentand treatment with hydrogen peroxide. We exposed cells to 200 J/m2UV, and then grew them for 4 or 7 hr before plating serial dilutions to test forviability (Figure 6A). To preventphoto-reactivation, the cells were grown in the dark prior to plating (Götz et al., 2007; Fröls et al., 2007). Viability wasessentially unaffected by 4-hr growth in the dark after UV irradiation; however,growth for 7 hr prior to plating resulted in survival dropping by two orders ofmagnitude. Despite the differences in viability, western blotting revealed nosignificant changes in the absolute or relative levels of primase catalytic andregulatory subunits (Figure 6A). Similarly,treatment of cells with 30 or 100 μM hydrogen peroxide for 4 or 7 hrimpacted on viability (Figures 6B and 6C),with severity of impact on cell viability scaling with concentration andexposure time. This ranged from an approximately 10-fold reduction in the numberof viable cells with a 4-hr treatment with 30 μM hydrogen peroxide tocomplete loss of viability with administration of 100 μM hydrogenperoxide. Regardless of the impact on viability, relative levels of primasesubunits were unaltered by H2O2 (Figures 6B and 6C). Thus, the effects of HU on levels ofPriL appear to be specific to treatment with this agent, not simply aconsequence of cell death. To determine whether HU could directly impact primasestability, we tested the effect of hydroxyurea on the purified primase complexin vitro. As can be seen in Figure 6D, toppanel, incubation of the heterotrimeric PriSLX complex with 5 or 10 mM HU for 4hr leads to enhanced precipitation of the complex. Compared to the water-treatedcontrol, we observe a 16- or 20-fold enrichment of primase in the insolublematerial for 5 and 10 mM HU, respectively. Incubation of recombinant MCMhelicase or DNA polymerase PolB1 with HU had no discernable impact on thesolubility of these proteins (Figure 6D,lower panels).

View Article: PubMed Central - PubMed

ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.