Réplication de l'ADN

Résumé

redresse=1.2|vignette La réplication de l'ADN, aussi appelée duplication de l'ADN ou synthèse de l'ADN, est le processus au cours duquel l'ADN est synthétisé. Ce mécanisme permet d'obtenir, à partir d'une molécule d'ADN, deux molécules identiques à la molécule initiale. L'ADN dupliqué sera par la suite divisé entre les deux cellules filles lors de la division cellulaire (mitose et méiose). Cela permet de maintenir l'information génétique et de produire deux cellules filles avec un matériel génétique identique.vignette|La mitose permet, grâce à la réplication de l'ADN, la formation de deux cellules filles strictement identiques génétiquement à la cellule mère. La réplication va commencer à des endroits précis: les origines de réplication. Les deux chaînes de l'ADN parental se séparent pour donner naissance à deux nouvelles molécules contenants, un brin parental et un brin nouvellement synthétisé, on qualifie ce processus de semi-conservateur. Un grand nombre de protéines interviennen

Source officielle

https://fr.wikipedia.org/wiki/R%C3%A9plication_de_l'ADN

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Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown.DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization(FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress.One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination.CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling.We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

Chargement

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

Adeno-associated virus (AAV) is a small DNA virus belonging to the family of the parvoviridae. AAV infects the human population, however no disease has been associated with this virus. On the contrary, AAV was reported to have oncosuppressive activities. The AAV genome contains two genes: the Rep gene encoding four non-structural proteins (Rep78, 68, 52 and 40) and the Cap gene encoding the three capsid proteins. Rep78 is implicated in transcription, replication and site-specific integration of the viral DNA. It has been shown to block the cell cycle in G1, G2 and S phase with a complete inhibition of DNA synthesis. To be able to induce such a strong cell cycle arrest, Rep78 affects several proteins implicated in the regulation of the cell cycle, such as the family of the cell division cycle 25 (Cdc25) phosphatases. Rep78 has been shown to inactivate Cdc25A by binding to it and preventing it from accessing its substrates. Cdc25B shares conserved domains with Cdc25A and moreover has an important role in cell cycle regulation, especially in giving the signal for entry into mitosis. Thus, in this work, the interaction between Cdc25B and Rep78 was examined in more detail. Rep78 was found to bind to Cdc25B and to alter its intracellular localisation. Cdc25B is nuclear, but has to migrate into the cytoplasm at the end of the G2 phase to activate proteins needed for the entry into mitosis. The expression of Rep78, which is nuclear, leads to the sequestration of Cdc25B in the nucleus. Moreover, Rep78 by interacting with Cdc25B not only changes its localisation, but also inhibits its phosphatase activity. Our model is that Rep78 interacts with Cdc25B and thus retains it in the nucleus. This sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis, thus contributing to the G2/M arrest induced by Rep78. Moreover, the inhibition of Cdc25B phosphatase activity by Rep78 may also contribute to the cell cycle block. Rep78 also interacts with a number of cellular proteins implicated in DNA replication, DNA repair or recombination. Interactions between Rep78 and mini-chromosome maintenance 3 (MCM3), DNA polymerases α and δ, proliferating cell nuclear antigen (PCNA), Rad50 and Rad51 were identified in this work as well as possible interactions between Rep78 and Ataxia telangiectasia mutated (ATM) or Ataxia telangiectasia and Rad3 related (ATR) proteins. These interactions may contribute to AAV genome replication as well as its integration into the host DNA.

EPFL2009

Chargement

Propagation of microorganisms is based on three fundamental processes: cell growth, DNA replication, and cell division. Although important for antibacterial drug development, these processes are poorly understood in Actinobacteria, a medically important phylum that includes Mycobacterium tuberculosis. Using microfluidic cultures and time-lapse microscopy, we studied single-cell growth, DNA replication, and cell division in the model organism Mycobacterium smegmatis.M. smegmatis is rod-shaped and grows by tip elongation in a biphasic manner due to a "new end take-off" (NETO) event. We find that pole elongation speed is increased and NETO occurs earlier in fast-growing cells than in slow-growing cells. As a consequence of variable timing of NETO, single-cell growth can be monophasic, biphasic, or triphasic. We propose that cells optimize pole growth speed and the timing of NETO to maximize their overall growth.We show that fast-growing cells initiate DNA replication earlier than slow-growing cells. We also find that single-cell growth speed is linked to cell-cycle progression, which is similar when comparing cells growing at the same speed under different conditions. We also report that multifork replication occurs when the time between DNA replication initiation events is shorter than the C period, which may occur even in slow-growing cells with interdivision times longer than the C period.Division site selection in many bacteria is governed by the nucleoid occlusion (Noc) and minicell (Min) systems. Mycobacteria do not encode homologs of these proteins and have no known mechanism for division site selection. We found that the DNA replisome and cell division ring colocalize and move together in a biphasic trajectory determined by the chromosome partitioning (Par) system. We propose a model in which Par-dependent movement of the replisome and division ring ultimately determines the site of cell division.Coordination of cell growth and division is required for cell size homeostasis. Three models of division control have been proposed: sizer (cells divide after reaching a critical size), timer (cells divide at a certain time after birth), and adder (cells add a specific amount of mass before dividing). We show that the single-cell growth model (exponential, linear, or bilinear) constrains the possible division control models. Thus, it is crucial to know the true model of single-cell growth in order to distinguish the true mechanism of cell size homeostasis.

EPFL2021

Chargement

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

EPFL2022

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

EPFL2009

EPFL2021