Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

A standardized method for the three-way differential staining of plant chromosomes and the scoring of SCEs per cell cycle

We have made use of 2 alternative methodologies to obtain 3-way differential staining (TWD) in third-mitosis (M₃) chromosomes of Allium cepa, which involve different uptakes of bromodeoxyuridine (BrdU) through 3 cell divisions, in order to evaluate the sister-chromatid exchanges (SCE) frequency on a per-cell-cycle basis.The main innovation introduced by us to the original protocols previously reported has been the use of 5-fluorodeoxyuridine (FdU) to inhibit endogenous thymidine (dT) synthesis. By using different [BrdU]: [dT] ratios in the presence of FdU the relative incorporation of BrdU into replicating DNA can be controlled.The use of 2 different approaches to obtain 3-way differentiation of sister chromatids allowed us to evaluate the role of BrdU in the induction of SCEs in our system. Both procedures rendered nearly 100% of M₃ chromosomes showing TWD. An additional advantage of our methodologies is their high degree of reproducibility.     

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with ³H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying ³H-thymidine (10^–6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10^–8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10^–6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.     

Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA

Ionizing radiation causes formation of thymine hydroperoxides in DNA. Their decomposition generates more stable products and active oxygen species which may oxidize other DNA bases. We have determined the effects of free and chelated metal ions on the degradation of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU). Two products were formed as analyzed by HPLC: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU). Sn(II) and Fe(II) caused instantaneous HPMdU degradation; Sn(II) generated only HMdU, whereas Fe(II) formed about equal amounts of both. Sn(IV) and Fe(III) were inactive. Cu(I), Cu(II), and Co(II) caused a time-dependent formation of both products, with FdU predominating. In the presence of Cu(I), Cu(II), and Fe(II), formate inhibited formation of HMdU but enhanced that of FdU. EDTA abolished Cu(I)-induced decomposition of HPMdU but only decreased that which was mediated by Cu(II). In contrast, EDTA enhanced the activity of Fe(III) with a time-dependent formation of FdU. EDTA and diethylenetriaminepentaacetic acid (DTPA) caused an instantaneous Fe(II)-mediated decomposition of HPMdU to FdU. Only desferal partially inhibited the activity of Fe(II), whereas the activities of Cu(I), Cu(II), and Fe(III) were blocked by desferal and DTPA. Possible mechanisms of HPMdU degradation by metal ions in the absence or presence of formate or chelators as well as formation of the .OH are discussed.     

Effect of Reversible Inhibition of Deoxyribonucleic Acid Synthesis on the Yeast Cell Cycle

Hydroxyurea (HU) preferentially inhibited deoxyribonucleic acid (DNA) replication and division in Saccharomyces cerevisiae. Growth, ribonucleic acid synthesis, and protein synthesis were less sensitive to this drug. Upon addition of HU, cells underwent one cycle of budding and the nuclei migrated into the necks between the mother cells and buds. Neither the nucleus nor the cells divided. Removal of HU allowed immediate resumption of DNA synthesis. Nuclear division, budding, and cell division occurred 1.5, 2, and 4 hr, respectively, after HU was removed. If protein synthesis was blocked at the time HU was removed, budding and cell division did not occur. These results were interpreted to indicate that HU prevents accumulation of the potential to initiate a new cell cycle.     

Is DNA synthesis required for the induction of differentiation in quiescent root cortical parenchyma?

Cortical parenchyma of pea roots normally does not divide nor differentiate as tracheary elements. After excision from the root these cells can be induced to undergo cell division followed by differentiation of tracheary elements in the presence of cytokinin. 5-Fluorodeoxyuridine prevents cell replication and cytodifferentiation. The thymidine analog, 5-bromodeoxyuridine, specifically prevents cytodifferentiation with little or no effect on the rate of cell replication. Thymidine can also prevent cytodifferentiation with little effect on cell replication. Thymidine reverses the effect of 5-bromodeoxyuridine (BrdU) or 5-fluorodeoxyuridine (FdU) when given simultaneous with or subsequent to either BrdU or FdU. BrdU given with FdU allows up to one round of cell replication but no cytodifferentiation. Differentiation is rarely observed unless the entire cell population has undergone the equivalent of two cell generations. These results are combined with those of other investigators to present an hypothesis that implicates DNA replication but not cytokinesis in the regulation of certain types of differentiation.     

Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.     

Synthesis and enzymatic characterization of P1-thio-P2-oxo trideoxynucleoside diphosphates having AZT, FdU, or dT at the 3'-position

Model compounds for oligonucleotide-prodrugs, P1-thio-P2-oxo-trideoxyribonucleoside diphosphates: d[G(s)C(o)X] and d[T(s)A(o)X] (X = AZT, FdU or dT) have been prepared, and their hydrolyses by snake venom phosphodiesterase and nuclease S1 are described.     

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization

The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by ³H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1–2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4–8 h. At 12–14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14–16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Adaptation of rat lateral gastrocnemius muscle motor units during hindlimb unloading

Contractile and fatigue-resistance properties of 71 lateral gastrocnemius muscle (LG) motor units (MU) following 14 days of hindlimb unloading (HU) were compared to those of 60 LG MU from control rats. The MU properties were assessed from isolated and stimulated individual motor axons. The MU were classified using standard criteria (shape of unfused tetani and fatigue resistance). The HU did not affect LG MU composition, but diminished the maximal tetanic tension (Po) of all MU types: P0 was significantly reduced by about 40% for the slow and fast-resistant MU, and by 18% for the fast-fatigable ones. The speed-related properties of fast-resistant MU became more similar to those of slower MU. The fatigue properties of MU were evaluated during a 5-min exercise test, using two fatigue indexes, FI2 and FI5, which expressed the relative capacity of MU to generate tension after 2 and 5 min, respectively. Results showed that 14 days of HU did not change the fatigue sensitivity of the LG MU. However, when F15 was compared to FI2, a greater decrease was observed after HU than in control conditions for the fast-resistant and fast-intermediate MU. It was concluded that a prolonged fatigue test may show changes in metabolic properties of muscle fibres during 14 days of HU. Specific adaptations of LG MU as well as comparisons with those of the soleus muscle under the same conditions are discussed.     

Pattern of chromosomal replication in synchronized lymphocytes

Summary The BrdUrd-Hoechst 33258-Giemsa technique was employed to study patterns of chromosomal replication in human lymphocytes synchronized by Methotrexate (MTX). It is proposed that in the presence of MTX, a major portion of the cell population is blocked in an advanced stage of the S-phase and not in the G₁/S border of the cell cycle. At this point, the replication of the chromosomal segments corresponding to the R-bands is terminated, and the replication of the G-bands and the inactive X-chromosomes is being initiated. The use of this method in the study of higher resolution patterns of chromosomal replications is proposed.     

DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.     

The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.     

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.     

Different Epidermal Cell Kinetic Effects of Hydroxyurea When Injected At Two Different Times of the Day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods—[³H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)—were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. the proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. the observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G₂ and in the mitotic rate. Hence the changes in G₂ and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

Background and Aims

Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis.

Methods

Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H₂O₂ production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation).

Key Results

Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H₂O₂, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants.

Conclusions

The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.     

NUCLEOCYTOPLASMIC RATIO REQUIREMENTS FOR THE INITIATION OF DNA REPLICATION AND FISSION IN TETRAHYMENA

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. ³H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G₁. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2–4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G₁ cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4× and the C continues to increase until just prior to recovery division when it also reaches a value 4× that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G₁. The elimination of G₁ (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.