Biomedical subjects

Aaron Bensimon

Publications and source records attributed to Aaron Bensimon.

7 recordsLinked to original sources

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↗

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↗