Publications by authors named "Guillaume Guilbaud"

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation.

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The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity.

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Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency.

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DNA can adopt non-B form structures that create significant blocks to DNA synthesis and seeking understanding of the mechanisms cells use to resolve such impediments continues to be a very active area of research. However, the ability to monitor the stalling of DNA synthesis at specific sites in the genome in living cells, of central importance to elucidating these mechanisms, poses a significant technical challenge. Replisome stalling is often transient with only a small fraction of events leading to detectable genetic changes, making traditional reporter assays insensitive to the stalling event per se.

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Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage.

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Background: Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. However, current studies of DNA synthesis through STRs are restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour and hampering the characterisation of the determinants of their abundance and stability in eukaryotic genomes.

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During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood.

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Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite.

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Article Synopsis
  • X-chromosome inactivation occurs early in development, with the paternal X-chromosome silenced by the blastocyst stage through epigenetic changes like H3K27me3.
  • This inactivation is reversed in the inner cell mass, but the exact processes behind the reactivation of the paternal X remain unclear.
  • Research using single-cell techniques indicates that genes reactivate at different rates based on their epigenetic status, with slowly reactivating genes showing more H3K27 marks, and UTX mutants further prolonging this process, highlighting the role of chromatin marks in gene reactivation.
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DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication.

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The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit.

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Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype.

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The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase.

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Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU) can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1(low) variants in dividing cells, which have lost the H3K4me3 present in untreated cells.

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G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures.

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The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics.

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Unlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle [1] and is incorporated into chromatin independent of replication [2]. Recently, H3.

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Article Synopsis
  • REV1-deficient chicken DT40 cells struggle to replicate DNA structures known as G quadruplexes (G4s), leading to instability in gene expression and chromatin marks.
  • A specific G4 motif was identified as the cause of epigenetic instability at the BU-1 locus, affecting gene expression despite being located far from the transcription start site.
  • The destabilizing effect of the G4 motif is linked to its position on the DNA strand, with significant loss of histone modifications occurring when the motif impacts the promoter region, emphasizing the importance of effective replication in maintaining gene expression.
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The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three domains of life. Here, we review our knowledge of genome organization into single and multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons show degenerate specificity and efficiency, allowing for complex regulation of origin firing time.

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In this protocol, we describe the use of the LastWave open-source signal-processing command language (http://perso.ens-lyon.fr/benjamin.

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The MCM proteins are best known for their role in DNA replication, MCM2-7 forming the replicative helicase. Now, two reports in this issue of Molecular Cell, Nishimura et al. (2012) and Lutzmann et al.

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In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew.

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Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.

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During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews.

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Neutral nucleotide substitutions occur at varying rates along genomes, and it remains a major issue to unravel the mechanisms that cause these variations and to analyze their evolutionary consequences. Here, we study the role of replication in the neutral substitution pattern. We obtained a high-resolution replication timing profile of the whole human genome by massively parallel sequencing of nascent BrdU-labeled replicating DNA.

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