of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G 1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO 2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200 -400 ppm O 2 or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.DNA replication in mammalian cells is subject to a fast acting regulation that depends on the O 2 tension in the cellular environment. This regulation results in inhibition of cellular replication when the concentration of O 2 falls below 0.1%. Regulation of cellular replication by O 2 has first been demonstrated for Ehrlich ascites cells (1-4). Further studies revealed that it is also valid for HeLa and CCRF cells (5), suggesting it to be a mechanism of general importance that adapts the cellular DNA replication to the supply of O 2 and other nutrients. This seems to be of particular significance during embryonic growth, wound healing, or tumor cell propagation. Inhibition of replication under hypoxia primarily affects replicon initiation. Additionally, the rate of DNA chain growth is frequently reduced. Readmission of O 2 after several hours of hypoxia reverses the suppression of DNA replication within a few minutes. This remarkably fast response suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.The molecular mechanism of the oxygen-dependent replication control is largely obscure. A reduction of the intracellular concentrations of deoxynucleoside triphosphates, e...

Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase d, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase a, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.Keywords: hypoxia; DNA unwinding; SV40; large T antigen; replicon initiation.DNA replication in mammalian cells is subject to a regulation, which depends on the O 2 tension in the cellular environment. This regulation results in inhibition of cellular replicon initiation when the concentration of O 2 falls below 0.1%. Re-oxygenation after several hours of hypoxia causes a burst of new initiations within a few minutes. So far, this regulatory phenomenon has been demonstrated for Ehrlichascites, HeLa and CCRF cells [1][2][3] and it may be a general mechanism, which adapts the cellular DNA replication to the supply of O 2 and other nutrients. This seems to be of particular significance during embryonic growth, wound healing or tumour cell propagation.The mechanism leading from re-oxygenation to replicon initiation is largely obscure. The remarkably fast resumption of initiations after re-oxygenation suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.O 2 -Dependent regulation of replicon initiation was also demonstrated for viral replication in simian virus 40 (SV40)-infected CV1 cells [4,5]. As the replication of SV40 is relatively well investigated, this virus seems to be well suited to exami...

We examined whether the fast release of replicon initiation after sudden O2 recovery of hypoxically incubated mammalian cells depends on kinase activity of Cdk2. We used a system based on starved/refed T24 cells elaborated previously for such investigations [van Betteraey‐Nikoleit M, Eisele KH, Stabenow D & Probst H (2003) Eur J Biochem270, 3880–3890]. Cells subjected to hypoxia concurrently with refeeding accumulate the G1 DNA content within 5–6 h. In this state they are ready to perform, within 1–2 min after O2 recovery, a burst of replicon initiations that marks the start of a synchronous S‐phase. We found that Cdk2 binds to the chromatin fraction within 4–6 h after refeeding with fresh medium, irrespective of whether the cells were incubated normoxically or hypoxically. However, inhibition of Cdk2 by olomoucine, roscovitine or the Cdk2/cyclin inhibitory peptide II had no influence on the synchronous burst of replicon initiations. Cdc6 and pRb, possible targets of Cdk2 phosphorylation, behaved differentially. Inhibition did not affect phosphorylation of Cdc6 after reoxygenation, whilst chromatin bound pRb remained hypophosphorylated beyond the initiation burst. Thus, neither Cdk2 activity, though present at the end of the hypoxic period, nor pRb phosphorylation are necessary for releasing the burst of replicon initiations upon oxygen recovery. Consequentially, Cdk2 dependent phosphorylation(s) cannot be a critical trigger of replicon initiation in response to reoxygenation after several hours of hypoxia, at least in the T24 cells studied.