TOPOREF · The Effect of DNA Topology on Eukaryotic Replication

The Effect of DNA Topology on Eukaryotic Replication

The replisome is the protein complex that carries out DNA replication. In eukaryotes, the core of the replisome is a helicase called CMG that unwinds the DNA double helix. Replication errors are a factor in the development of cancer. New insights into eukaryotic replication could contribute to novel treatments or diagnostic tools for cancer that would be of great social and economic value. Over the past 30 years, our knowledge of the yeast replisome has grown to encompass all its protein components and a growing list of factors that mediate its interaction with chromatin. The order of assembly of the replisome is well understood, as are the mechanisms by which it is kept in sync with the cell cycle. However, the physical processes that activate the replisome and the dynamics of its interaction with the DNA substrate remain under active investigation. On a higher level, every aspect of replication hinges on the physical manipulation of DNA. Accordingly, replication must involve the controlled application, monitoring, and relaxation of torque on that molecule. In this proposal, I aim to address the relationship between DNA torque and twist during replication. Project A concerns the assembly of the replisome. At what point in CMG assembly does melting of the DNA double helix occur? What are the accompanying timescales and roles of critical factors? What is the effect of supercoiling and tension in the DNA? Project B picks up the kinetics of the advancing replisome. My goal is to characterize how the rate and pause dynamics of the replisome depend on torque on the DNA as well as on critical firing factors. Project C concerns measurement of the torque required to stall the replisome. If the replisome advances far enough in the absence of certain accessory factors, the torque that builds up in front of the replisome will eventually stall the replicative helicase when it can no longer generate the force needed to melt the strands. Project D. The basic subunit of chromatin is the wrapping of DNA around nucleosomes. The presence of nucleosomes decreases the torsional stiffness of DNA, suggesting that they are able to absorb torque generated by the replisome by flipping their orientation. By comparing the stalling dynamics of replisomes in the presence and absence of nucleosomes, I will determine whether nucleosomes allow the replisome to advance further before stalling.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз