Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.

Single linear DNA molecules were bound at multiple sites at one extremity to a treated glass cover slip and at the other to a magnetic bead. The DNA was therefore torsionally constrained. A magnetic field was used to rotate the beads and thus to coil and pull the DNA. The stretching force was determined by analysis of the Brownian fluctuations of the bead. Here the elastic behavior of individual lambda DNA molecules over- and underwound by up to 500 turns was studied. A sharp transition was discovered from a low to a high extension state at a force of approximately 0.45 piconewtons for underwound molecules and at a force of approximately 3 piconewtons for overwound ones. These transitions, probably reflecting the formation of alternative structures in stretched coiled DNA molecules, might be relevant for DNA transcription and replication.

SUMMARY Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.

In a process called "molecular combining," DNA molecules attached at one end to a solid surface were extended and aligned by a receding air-water interface and left to dry on the surface. Molecular combing was observed to extend the length of the bacteriophage lambda DNA molecule to 21.5 +/- 0.5 micrometers (unextended length, 16.2 micrometers). With the combing process, it was possible to (i) extend a chromosomal Escherichia coli DNA fragment (10(6) base pairs) and (ii) detect a minute quantity of DNA (10(3) molecules). These results open the way for a faster physical mapping of the genome and for the detection of small quantities of target DNA from a population of molecules.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

A detailed experimental and theoretical analysis of the alignment of grafted DNA molecules by a moving meniscus is presented. The existence and extent of the stretching (up to 2.14 times the unstretched length) depends critically on the properties of the surface. Molecules grafted at both ends exhibit a looplike shape which is scale invariant. An elastic model of this process, which we have called molecular combing, is introduced which (a) yields the extension force on various surfaces, (b) yields a value for the tensile strength of DNA, 476 6 84 pN, and (c) describes the shape of the loops with no fitting parameters.

Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed.

Glutathione S-transferase (GST) Ya subunit gene expression is induced in mammalian tissues by two types of chemical agents: (i) planar aromatic compounds (e.g., 3-methylcholanthrene, g3-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and (i) electrophiles (e.g., trans-4-phenyl-3-buten-2-one and dimethyl fumarate) or compounds easily oxidized to electrophiles (e.g., tert-butylhydroquinone).To study the mechanism of this induction, we have introduced deletions in the 5' flanking region of a mouse GST Ya subunit gene, fused it to the coding sequence for chloramphenicol acetyltransferase (CAT) To elucidate the mechanisms of regulation of GST Ya subunit gene expression by the two types of inducers, we have previously isolated a mouse GST Ya gene (7, 8) and have shown the presence of xenobiotic responsive elements in the 5' flanking region (9). In the present study, we demonstrate that this region contains between nucleotides -754 and -713 an inducible element that activates Ya gene transcription in cis in response to a variety of inducers such as 3-methylcholanthrene, p-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tert-butylhydroquinone, and trans4phenyl-3-buten-2-one. These results raise the question of the mechanisms proposed for the regulation of GST gene expression by planar aromatic and electrophilic inducers (4,5). Our results show that, in order to function as Ya gene inducers, the planar aromatics have to be metabolized by the cytochrome P1-450 system into electrophilic compounds. In this paper we bring evidence that the inducible expression of GST Ya subunit gene is controlled by a single electrophile-responsive element (EpRE), which is activated exclusively by an electrophilic signal. Protein (Weizmann Institute). The cell lines were propagated as described (9). Nuclear and cytosolic extracts were prepared from the cell lines as described by Dignam et al. (10), and DNase I protection patterns (footprint assays) have been described (9).Transfections. The plasmid constructions containing fragments of the 5' flanking region of the GST Ya subunit gene Abbreviations: GST, glutathione S-transferase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CAT, chloramphenicol acetyltransferase; EpRE, electrophile-responsive element. *Phase I xenobiotic metabolizing enzymes (e.g., cytochrome P1-450) introduce by oxidation or reduction functional groups into chemical compounds. 6258The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.