Accumulation of short DNA fragments in hydroxyurea treated mouse L-cells

Martin, Roger F.; Radford, Ian R.; Pardee, Marshall

doi:10.1016/0006-291x(77)91368-7

Cited by 27 publications

(11 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6F). This assumption is supported by several lines of evidence that hydroxyurea treatment results in decreased purine dNTP pools, increased UV-induced single strand breaks (Snyder, 1984;Hunting and Dresler, 1985), and retarded lagging strand gap-filling DNA synthesis (Martin et al, 1977). Our findings also demonstrate that treatment with triapine, which is 1000 times more potent than hydroxyurea, causes sustained and pronounced depletion of dATP and dGTP, whereas dCTP and dTTP are less affected and rapidly rebound to normal levels (Supplemental Fig.…”

Section: Tablesupporting

confidence: 84%

Reduced Level of Ribonucleotide Reductase R2 Subunits Increases Dependence on Homologous Recombination Repair of Cisplatin-Induced DNA Damage

Lin

Lee²,

Lin³

et al. 2011

Mol Pharmacol

View full text Add to dashboard Cite

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) required for replicative and repair DNA synthesis. Mammalian RNR is a heteromeric enzyme consisting primarily of R1 and R2 subunits during the S phase of the cell cycle. We have shown previously that the presence of excess R2 subunits protects p53-deficient human colon cancer cells from cisplatin-induced DNA damage and replication stress. However, the mode of DNA repair influenced by changes in the level of the R2 subunit remained to be defined. In the present study, we demonstrated that depletion of BRCA1, an important factor of homologous recombination repair (HRR), preferentially sensitized stable R2-knockdown p53(Ϫ/Ϫ) HCT116 cells to the cytotoxicity of cisplatin and ␥-H2AX induction. In accord with this finding, these R2-knockdown cells exhibited increased dependence on HRR, as evidenced by elevated levels of cisplatin-induced Rad51 foci and sister chromatid exchange frequency. Furthermore, stable knockdown of the R2 subunit also led to decreased cisplatin-induced gap-filling synthesis in nucleotide excision repair (NER) and a reduced dATP level in the G 2 /M phase of the cell cycle. These results suggest that an increased level of the R2 subunit extends the availability of dATP in the G 2 /M phase to promote the repair of NER-mediated singlestrand gaps that are otherwise converted into double-strand breaks in the subsequent S phase. We propose that HRR becomes important for recovery from cisplatin-DNA lesions when the postexcision process of NER is restrained by reduced levels of the R2 subunit and dATP in p53-deficient cancer cells.

show abstract

Section: Tablesupporting

confidence: 84%

Reduced Level of Ribonucleotide Reductase R2 Subunits Increases Dependence on Homologous Recombination Repair of Cisplatin-Induced DNA Damage

Lin

Lee²,

Lin³

et al. 2011

Mol Pharmacol

View full text Add to dashboard Cite

show abstract

“…Fig. 4A and B); the size distribution of labeled strands resembled that reported for cellular DNA synthesis inhibited in vivo by HU (16,25,29). Whereas the sedimentation pattern of the control lysate reached a steady state after 5 min, the size of the products in the HU-influenced nuclei slightly increased between 5 and 15 min.…”

Section: Resultssupporting

confidence: 78%

“…It suppresses the polymerization of new strands almost completely and causes the accumulation of short DNA fragments that cannot be further elongated (16,21,24,25,29). Repair synthesis is much less affected by the drug (34).…”

mentioning

confidence: 99%

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Wawra

Wintersberger

1983

Mol. Cell. Biol.

View full text Add to dashboard Cite

Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the Gl/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.Hydroxyurea (HU) is a specific inhibitor of DNA replication (34). It suppresses the polymerization of new strands almost completely and causes the accumulation of short DNA fragments that cannot be further elongated (16,21,24,25,29). Repair synthesis is much less affected by the drug (34). It has generally been assumed that the block of the conversion of ribonucleotides to deoxyribonucleotides via the inhibition of the enzyme ribonucleoside diphosphate reductase is responsible for these effects of HU (17,18,20). On the other hand, the difference in the response of replicative versus repair synthesis to HU is not obvious; the substrate affinities of the known DNA polymerases do not vary enough to provide a satisfactory rationale (4, 6). Moreover, the proposed mechanism cannot explain why HU-blocked cells do not stop at the Gl/S boundary but start replication and accumulate in the early S phase (10, 36).The observation that, in a cell-free DNAsynthesizing system obtained from HU-treated cells, the inhibition was still partly effective although all triphosphates were present in theincubation mixture led us to reconsider the effect of HU. This phenomenon was specific for HU and could not be observed with fluorodeoxyuridine (FUdR), another substance which interferes with the precursor pool. Hence, it is unlikely that the observed lasting inhibition is due to the interference of HU with the biosynthesis of precursors. A more detailed investigation of this effect of HU is the subject of this report. (41) and grown in plastic petri dishes at 37°C in a water-saturated atmosphere with 7.5% C02, using DME medium containing 10% calf serum. After reaching confluence, cells stopped division, and they were usually us...

show abstract

“…A similar phenomenon is observed in cells treated with hydroxyurea, which inhibits the ribonucleoside diphosphate reductase (246), affects chain elongation (247), and causes accumulation of newly replicated, 7S single strand fragments (248)(249)(250). Temporary inhibition of semiconservative rep-EUKARYOTIC DNA REPLICATION 29 1 lication by this drug (247,251) results in arrest of mammalian cells (135,244,(247)(248)(249)(250)(251)(252) and yeast (253,254) early in S phase.…”

Section: Studies With Inhibitors Of Dna Synthesismentioning

confidence: 64%