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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

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).

Ahuja A.K.,University of Zurich | Jodkowska K.,DNA Replication Group | Teloni F.,University of Zurich | Bizard A.H.,Copenhagen University | And 7 more authors.
Nature Communications | Year: 2016

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.

Bua S.,DNA Replication Group | Bua S.,Institute Pasteur Paris | Sotiropoulou P.,Free University of Colombia | Sgarlata C.,DNA Replication Group | And 10 more authors.
Cell Cycle | Year: 2015

Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice. © 2015 Taylor & Francis Group, LLC.

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.

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