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News Article | May 4, 2017
Site: www.biosciencetechnology.com

DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explained Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explained Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", said Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body.


News Article | May 3, 2017
Site: www.eurekalert.org

Simultaneous over-expression of two proteins provokes DNA re-replication in some cell types. This phenomenon can lead to cell malignancy but also might be used to attack cancer cells DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explains Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explains Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", says Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body.


News Article | May 3, 2017
Site: phys.org

DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explains Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explains Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", says Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body. Explore further: DNA replication protein Cdt1 also has a role in mitosis, cancer


Sanchez-Berrondo J.,Macromolecular Crystallography Group | Mesa P.,Macromolecular Crystallography Group | Ibarra A.,DNA Replication Group | Ibarra A.,Salk Institute for Biological Studies | And 3 more authors.
Nucleic Acids Research | Year: 2012

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus. © 2011 The Author(s).


Guillou E.,DNA Replication Group | Guillou E.,Laboratoire Of Biologie Moleculaire Eucaryote | Ibarra A.,DNA Replication Group | Coulon V.,Montpellier University | And 8 more authors.
Genes and Development | Year: 2010

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. © 2010 by Cold Spring Harbor Laboratory Press.


Aparicio T.,DNA Replication Group | Aparicio T.,Columbia University Medical Center | Megias D.,Confocal Microscopy Unit | Mendez J.,DNA Replication Group
Chromosoma | Year: 2012

In mammalian cells, DNA synthesis takes place at defined nuclear structures termed "replication foci" (RF) that follow the same order of activation in each cell cycle. Intriguingly, immunofluorescence studies have failed to visualize the DNA helicase minichromosome maintenance (MCM) at RF, raising doubts about its physical presence at the sites of DNA synthesis. We have revisited this paradox by pulse-labeling RF during the S phase and analyzing the localization of MCM at labeled DNA in the following cell cycle. Using high-throughput confocal microscopy, we provide direct evidence that MCM proteins concentrate in G1 at the chromosome structures bound to become RF in the S phase. Upon initiation of DNA synthesis, an active "MCM eviction" mechanism contributes to reduce the excess of DNA helicases at RF. Most MCM complexes are released from chromatin, except for a small but detectable fraction that remains at the forks during the S phase, as expected for a replicative helicase. © Springer-Verlag 2012.


Lopez-Contreras A.J.,Genomic Instability Group | Ruppen I.,Proteomics Unit | Nieto-Soler M.,Genomic Instability Group | Murga M.,Genomic Instability Group | And 8 more authors.
Cell Reports | Year: 2013

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance. © 2013 The Authors.


Trakala M.,Cell Division and Cancer Group | Rodriguez-Acebes S.,DNA Replication Group | Maroto M.,Cell Division and Cancer Group | Symonds C.E.,Experimental Oncology Group | And 5 more authors.
Developmental Cell | Year: 2015

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process inwhich megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms invivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells. © 2015 Elsevier Inc.


Munoz S.,DNA Replication Group | Mendez J.,DNA Replication Group
Chromosoma | Year: 2016

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of “replicative stress” (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS. © 2016 Springer-Verlag Berlin Heidelberg


PubMed | DNA Replication Group
Type: | Journal: Chromosoma | Year: 2016

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of replicative stress (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

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