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Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase

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.


Related in: MedlinePlus

Molecular Consequences of Treatment of S. solfataricus withHU(A) Western blot analysis of the levels of a variety of replication and celldivision-associated proteins in Sulfolobus following treatmentwith 10 mM HU for 7 hr. The anti-WhiP antisera detects full-length protein andan additional truncated form. Loading for all panels was confirmed by westernblotting for the general transcription factor, TBP. A single representative TBPpanel is shown.(B) Flow cytometry profile confirming the cell cycle arrest upon HU treatment forthe cells used in (A).(C) Western blot analyses of the effect of 7 hr exposure to the indicatedconcentrations of HU on the levels of Orc1-1, Orc1-3, and primase subunits. Flowcytometry profiles of the cells following treatment are shown on the right. TBPserves as a loading control in this and the subsequent panel.(D) Effect of varying the time of exposure to 10 mM HU on the levels of Orc1-1,Orc1-3, and primase subunits. Flow cytometry profiles of the treated cells areshown to the right of the western blot images.
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Figure 2: Molecular Consequences of Treatment of S. solfataricus withHU(A) Western blot analysis of the levels of a variety of replication and celldivision-associated proteins in Sulfolobus following treatmentwith 10 mM HU for 7 hr. The anti-WhiP antisera detects full-length protein andan additional truncated form. Loading for all panels was confirmed by westernblotting for the general transcription factor, TBP. A single representative TBPpanel is shown.(B) Flow cytometry profile confirming the cell cycle arrest upon HU treatment forthe cells used in (A).(C) Western blot analyses of the effect of 7 hr exposure to the indicatedconcentrations of HU on the levels of Orc1-1, Orc1-3, and primase subunits. Flowcytometry profiles of the cells following treatment are shown on the right. TBPserves as a loading control in this and the subsequent panel.(D) Effect of varying the time of exposure to 10 mM HU on the levels of Orc1-1,Orc1-3, and primase subunits. Flow cytometry profiles of the treated cells areshown to the right of the western blot images.

Mentions: Given the perturbation to the cell cycle profile of the populationfollowing HU treatment, we profiled relative levels of a number of DNAreplication, DNA repair, and cell division-associated proteins following HUtreatment. Initially, we tested the effect of 7-hr exposure to 10 mM HU (Figures 2A and 2B). Many of the proteins thatwe tested showed no significant changes in level following HU treatment (Figure 2A). These included replicationfork-associated proteins such as the single-strand DNA binding protein (SSB);the replicative helicase MCM; the MCM-interacting factor Gins23; sliding clampsubunits PCNA1, 2, and 3; DNA ligase; replicative DNA polymerase polB1; and theclamp loader RFC. In addition, levels of WhiP, a homolog of the eukaryalpre-replicative complex protein Cdt1 that acts as the initiator protein thatgoverns oriC3 (Robinson andBell, 2007; Samson et al.,2013); the chromatin protein Alba (Bell et al., 2002); the candidate fork regression helicase Hel308;and the recombinase RadA (the RAD51/RecA ortholog) were unaltered in theirlevels following treatment with HU.


Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase
Molecular Consequences of Treatment of S. solfataricus withHU(A) Western blot analysis of the levels of a variety of replication and celldivision-associated proteins in Sulfolobus following treatmentwith 10 mM HU for 7 hr. The anti-WhiP antisera detects full-length protein andan additional truncated form. Loading for all panels was confirmed by westernblotting for the general transcription factor, TBP. A single representative TBPpanel is shown.(B) Flow cytometry profile confirming the cell cycle arrest upon HU treatment forthe cells used in (A).(C) Western blot analyses of the effect of 7 hr exposure to the indicatedconcentrations of HU on the levels of Orc1-1, Orc1-3, and primase subunits. Flowcytometry profiles of the cells following treatment are shown on the right. TBPserves as a loading control in this and the subsequent panel.(D) Effect of varying the time of exposure to 10 mM HU on the levels of Orc1-1,Orc1-3, and primase subunits. Flow cytometry profiles of the treated cells areshown to the right of the western blot images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134839&req=5

Figure 2: Molecular Consequences of Treatment of S. solfataricus withHU(A) Western blot analysis of the levels of a variety of replication and celldivision-associated proteins in Sulfolobus following treatmentwith 10 mM HU for 7 hr. The anti-WhiP antisera detects full-length protein andan additional truncated form. Loading for all panels was confirmed by westernblotting for the general transcription factor, TBP. A single representative TBPpanel is shown.(B) Flow cytometry profile confirming the cell cycle arrest upon HU treatment forthe cells used in (A).(C) Western blot analyses of the effect of 7 hr exposure to the indicatedconcentrations of HU on the levels of Orc1-1, Orc1-3, and primase subunits. Flowcytometry profiles of the cells following treatment are shown on the right. TBPserves as a loading control in this and the subsequent panel.(D) Effect of varying the time of exposure to 10 mM HU on the levels of Orc1-1,Orc1-3, and primase subunits. Flow cytometry profiles of the treated cells areshown to the right of the western blot images.
Mentions: Given the perturbation to the cell cycle profile of the populationfollowing HU treatment, we profiled relative levels of a number of DNAreplication, DNA repair, and cell division-associated proteins following HUtreatment. Initially, we tested the effect of 7-hr exposure to 10 mM HU (Figures 2A and 2B). Many of the proteins thatwe tested showed no significant changes in level following HU treatment (Figure 2A). These included replicationfork-associated proteins such as the single-strand DNA binding protein (SSB);the replicative helicase MCM; the MCM-interacting factor Gins23; sliding clampsubunits PCNA1, 2, and 3; DNA ligase; replicative DNA polymerase polB1; and theclamp loader RFC. In addition, levels of WhiP, a homolog of the eukaryalpre-replicative complex protein Cdt1 that acts as the initiator protein thatgoverns oriC3 (Robinson andBell, 2007; Samson et al.,2013); the chromatin protein Alba (Bell et al., 2002); the candidate fork regression helicase Hel308;and the recombinase RadA (the RAD51/RecA ortholog) were unaltered in theirlevels following treatment with HU.

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.


Related in: MedlinePlus