Biomedical subjects

Aaron Bensimon

Publications and source records attributed to Aaron Bensimon.

17 recordsLinked to original sources

PubMed2006

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.

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.

Animals↗

PubMed2006

DNA replication origin interference increases the spacing between initiation events in human cells.

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.

Base Pairing↗

PubMed2006

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

Blotting, Southern↗

PubMed2005

DNA replication origins fire stochastically in fission yeast.

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

DNA Replication↗

PubMed2005

Human ribosomal RNA gene arrays display a broad range of palindromic structures.

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.

Blotting, Southern↗

PubMed2005

Genomic organization of amplified MYC genes suggests distinct mechanisms of amplification in tumorigenesis.

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer-derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge-type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer-derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.

Female↗

PubMed2005

DNA replication origin plasticity and perturbed fork progression in human inverted repeats.

The stability of metazoan genomes during their duplication depends on the spatiotemporal activation of origins and the progression of forks. Human rRNA genes represent a unique challenge to DNA replication since a large proportion of them exist as noncanonical palindromes in addition to canonical tandem repeats. Whether origin usage and/or fork elongation can cope with the variable structure of these genes is unknown. By analyzing single combed DNA molecules from HeLa cells, we studied the rRNA gene replication program according to the organization of canonical versus noncanonical rRNA genes. Origin positioning, spacing, and timing were not affected by the underlying rRNA gene physical structure. Conversely, fork arrest, both temporary and permanent, occurred more frequently when rRNA gene palindromes were encountered. These findings reveal that while initiation mechanisms are flexible enough to adapt to an rRNA gene structure of any arrangement, palindromes represent obstacles to fork progression, which is a likely source of genomic instability.

DNA Replication↗

PubMed2004

Persistence length of chromatin determines origin spacing in Xenopus early-embryo DNA replication: quantitative comparisons between theory and experiment.

In Xenopus early embryos, replication origins neither require specific DNA sequences nor is there an efficient S/M checkpoint, even though the whole genome (3 billion bases) is completely duplicated within 10-20 minutes. This leads to the "random-completion problem" of DNA replication in embryos, where one needs to find a mechanism that ensures complete, faithful, timely reproduction of the genome without any sequence dependence of replication origins. We analyze recent DNA replication data in Xenopus laevis egg extracts and find discrepancies with models where replication origins are distributed independently of chromatin structure. Motivated by these discrepancies, we have investigated the role that chromatin looping may play in DNA replication. We find that the loop-size distribution predicted from a wormlike-chain model of chromatin can account for the spatial distribution of replication origins in this system quantitatively. Together with earlier findings of increasing frequency of origin firings, our results can explain the random-completion problem. The agreement between experimental data (molecular combing) and theoretical predictions suggests that the intrinsic stiffness of chromatin loops plays a fundamental biological role in DNA replication in early-embryo Xenopus in regulating the origin spacing.

Animals↗

PubMed2003

Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing.

Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.

AMP Deaminase↗

PubMed2003

Characterisation of a 161 kb deletion extending from the NBR1 to the BRCA1 genes in a French breast-ovarian cancer family.

A large germline deletion removing exons 1 to 22 of the BRCA1 gene has been previously detected using quantitative PCR based methods (QMPSF and real time PCR gene dosage assay) in a woman affected with breast and ovarian cancer. Here, we report its characterisation by using colour bar code on combed DNA of the BRCA1 region. The 5' boundary is located in a Alu Y sequence in NBR1 intron 18 whereas the 3' boundary is located in a Alu Sc sequence in BRCA1 intron 22. This 161 kb deletion encompassing the NBR1, PsiBRCA1, NBR2 and BRCA1 genes is the largest BRCA1 deletion reported so far. No specific phenotype was associated with the hemizygosity of these four genes.

BRCA1 Protein↗

PubMed2003

Single DNA molecule analysis: applications of molecular combing.

Dynamic changes to the genomic structure and to the DNA replication programme are important determinants of normal and abnormal cell development. To understand these changes and how they vary from cell to cell, single DNA molecules from both normal and abnormal cell populations must be examined and compared. Physical characterisation of single genomes at the kilobase level of resolution over large genomic regions is possible with molecular combing technology. An array of combed single DNA molecules is prepared by stretching molecules attached by their extremities to a silanised glass surface with a receding air-water meniscus. By performing fluorescent hybridisation on combed DNA, genomic probe position can be directly visualised, providing a means to construct physical maps and detect micro-rearrangements. Single-molecule DNA replication can also be monitored through fluorescent detection of incorporated nucleotide analogues on combed DNA molecules. These and other single-molecule applications of molecular combing are discussed in this paper and future developments of the technology are considered.

DNA↗

PubMed2002

Significant contribution of large BRCA1 gene rearrangements in 120 French breast and ovarian cancer families.

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast-ovarian cancers. However, BRCA1 germline mutations are found much less frequently than expected, especially as standard PCR-based mutation detection approaches focus on point and small gene alterations. In order to estimate the contribution of large gene rearrangements to the BRCA1 mutation spectrum, we have extensively analysed a series of 120 French breast-ovarian cancer cases. Thirty-eight were previously found carrier of a BRCA1 point mutation, 14 of a BRCA2 point mutation and one case has previously been reported as carrier of a large BRCA1 deletion. The remaining 67 cases were studied using the BRCA1 bar code approach on combed DNA which allows a panoramic view of the BRCA1 region. Three additional rearrangements were detected: a recurrent 23.8 kb deletion of exons 8-13, a 17.2 kb duplication of exons 3-8 and a 8.6 kb duplication of exons 18-20. Thus, in our series, BRCA1 large rearrangements accounted for 3.3% (4/120) of breast-ovarian cancer cases and 9.5% (4/42) of the BRCA1 gene mutation spectrum, suggesting that their screening is an important step that should be now systematically included in genetic testing surveys.

Adult↗

PubMed2002

Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus.

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of approximately 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2 Delta mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.

DNA Replication↗

PubMed2002

Kinetic model of DNA replication in eukaryotic organisms.

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

Animals↗

PubMed2002

Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae.

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Amino Acid Sequence↗

PubMed2002

A combinatorial approach for fast, high-resolution mapping.

High-resolution physical maps can be used as a scaffold for several subsequent studies, such as sequencing projects and positional cloning of disease genes and genetic elements that regulate gene expression. Here we describe a method for fast, high-resolution physical mapping on stretched DNA molecules, based on a combinatorial multi-FISH approach. Fluorescent labels are assigned to a binary code and probes are identified by a binary tag according to their labeling. To validate the approach, we have mapped eight probes covering a region of about 300 kb on human chromosome 11 with three hybridization assays. This approach enables one to determine the structural organization of a large region by means of the order of its clones, without ambiguities. The structure established in a control cell constitutes a reference for further studies, to detect rearrangements displayed by disease cells and to find differences shown by different cell types and organisms.

Humans↗

PubMed2002

Combing the genome for genomic instability.

Genomic instability is one of the major features of cancer cells. The clinical phenotypes associated with several human diseases have been linked to recurrent DNA rearrangements and dysfunction of DNA replication processes that involve unstable genomic regions. Analysis of these rearrangements, which are frequently submicroscopic and can lead to loss or gain of dosage-sensitive genes or gene disruption, requires the development of sensitive, high-resolution techniques. This will lead to a better understanding of the mechanisms underlying genome instability and a greater awareness of the role of chromosomal rearrangements in disease. A new technology that involves molecular combing, a method that permits straightening and aligning molecules of genomic DNA, should make possible a detailed analysis of genomic events at the level of single DNA molecules. Such a single molecule approach could help to elucidate important properties that are masked in bulk studies.

Cell Transformation, Neoplastic↗