59 research outputs found
Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading
No full textGetting loaded—make mine a double!
Chromosomal DNA replication initiates bidirectionally by loading two ring-shaped helicases onto DNA in opposite orientations. How this symmetry is achieved has been puzzling because replication initiation sites contain only one essential binding site for the initiator, the origin recognition complex (ORC). Coster and Diffley now show that both helicases are loaded by a similar mechanism. Efficient loading requires binding of two ORC complexes to two ORC binding sites in opposite orientations. Natural origins were found to be partially symmetrical, containing functionally relevant secondary ORC sites. Sites can be flexibly spaced, but introducing an intervening “roadblock” prevented loading, suggesting that individual helicases translocate toward each other on DNA to form a stable double ring.
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A single-molecule approach to understanding sister chromatid cohesion establishment
No full textSister chromatid cohesion is established by the cohesin complex during DNA replication in S phase. This process requires cohesin to transition from binding to one DNA strand to co-entrapping the two newly synthesised sister chromatids. Passage of the eukaryotic replisome through the ring-shaped cohesin complex has been proposed as a fail-safe mechanism to ensure that cohesin entraps the two sister chromatids, enabling their faithful segregation during cell division. However, whether replisomes can indeed pass through cohesin remains unknown, and the details of replisome-cohesin encounters are poorly understood.
Here, I use single molecule fluorescence microscopy to directly visualise encounters between biochemically reconstituted DNA replication forks and cohesin complexes. On a linear forked DNA, I find that the translocating replicative CMG helicase acts as an obstacle to cohesin. The obstacle is overcome by cohesin at low frequencies, which increase in presence of added replisome components with known sister chromatid cohesion establishment functions. When lateral cohesin diffusion is prevented, CMG passage, during which cohesin retains topological DNA entrapment, becomes the predominant outcome. Finally, I find that the likelihood of passage also increases when an active replisome encounters cohesin, resulting in successful cohesion establishment between the two replication products. Interestingly, when cohesin’s ability to diffuse on DNA is blocked, replication fork passage is efficiently achieved independently of cohesion establishment factors.
My findings resolve a long-held puzzle by visualising live sister chromatid cohesion establishment as the replisome passes the cohesin ring, providing new insights into the molecular mechanisms of sister chromatid cohesion establishment.Open Acces
Quality control in the initiation of eukaryotic DNA replication
No full textOrigins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.</jats:p
Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment
No full textEukaryotic cells respond to DNA damage by activating checkpoint signalling pathways. Checkpoint signals are transduced by a protein kinase cascade that also requires non-kinase mediator proteins. One such mediator is the Saccharomyces cerevisiae Dpb11 protein, which binds to and activates the apical checkpoint kinase, Mec1. Here, we show that a ternary complex of Dpb11, Mec1 and another key mediator protein Rad9 is required for efficient Rad9 phosphorylation by Mec1 in vitro, and for checkpoint activation in vivo. Phosphorylation of Rad9 by cyclindependent kinase (CDK) on two key residues generates a binding site for tandem BRCT repeats of Dpb11, and is thereby required for Rad9 recruitment into the ternary complex. Checkpoint signalling via Dpb11, therefore, does not efficiently occur during G1 phase when CDK is inactive. Thus, Dpb11 coordinates checkpoint signal transduction both temporally and spatially, ensuring the initiator kinase is specifically activated in proximity of one of its critical substrates
Break dosage, cell cycle stage and DNA replication influence DNA double strand break response
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Multiple DNA repair pathways contribute to cell lethality in checkpoint mutants
No full textTrabajo presentado al Midterm Review Meeting; Checkpoints, DNA Damage Response and Cancer, celebrado en Milán el 3 de noviembre de 2005.The checkpoint kinases Mec1 and Rad53 play an essential role in stabilizing stalled
DNA replication forks. We have initiated a genetic screen to identify mutants that
render checkpoint-defective yeast cells more resistant to fork-stalling agents (e.g.
hydroxyurea, MMS, etc). We have screened the entire library for mutants with
heightened resistance to hydroxyurea when the checkpoint has been compromised with
caffeine. From this, we have identified mutants in several repair pathways, and
therefore, we decided to check for caffeine plus hydroxyurea resistance in several
members of the main repair pathways. Our conclusions indicate that different repair
pathways contribute to lethality in checkpoint mutants.
We have also found that there is a significant Increase in HU-resistance when
combining different repair mutants in rad53-null background. However, none of these
multiple mutants is able to rescue viability completely, indicating that there are other
requirements to maintain fork stability in the absence of rad53.Peer reviewe
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