Single-molecule microscopy of blocked replication forks – MicroRep

The technique used in this work is particularly powerful and provides original insight. It allows the observation of single or few molecules, allows determining their precise amount when they act, and measuring their kinetic of interaction with DNA or with other proteins. By varying the growth conditions or the genetic background we can identify the elements that control their action.

Our first results show that multi-molecular replication complexes, considered as very tightly bound, are dissociated when replication is arrested. Proteins involved in genome stability are involved in this dissociation, in agreement with the idea that replication arrest is directly linked to genome rearrangements. These results have been published in Molecular Cell, a high rank international journal, in February 2013.
A second topic is the subject of present research. Several proteins invovled in replication restart have been shown to interact with replisome components. Whether they interact in growing cells with functional replication proteins or only when replication is blocked is an open question. Several proteins that play a role in homologous recombination (DNA repair by exchanges with intact DNA sequences) or that play a role in replication restart have been fused to fluorescent proteins. We have observed that these proteins interact with functional replisomes in normally growing cells, which modify our view of the structure of the replisome.

The presence on functional replication forks of proteins that will only be useful in case of replication arrest shows the importance of preserving arrested replication forks for cellular viability.

We have published that after replication arrest the replication machinery falls off DNA little by little, and that homologous recombination enzymes play a role in this disassembly in the international journal Molecular Cell.
Lia, G., Rigato, A., Long, E., Chagneau, C., LeMasson, M., Allemand, JF., Michel, B. (2013) RecA-promoted, RecFOR-independent progressive disassembly of replication forks stalled by helicase inactivation. Mol Cell, 49 1-11.

Replication defects can have dramatic consequences and are a recognized source of genomic instability in all organisms. Our aim is to describe and understand the reactions that take place at inactivated replication forks in the model organism Escherichia coli. We will use a novel single-molecule microscopy technique which allows the detection of as few as one fluorescent molecule, provided that it is DNA-bound. We will thus be able to observe specifically the enzymes at work in living cells, first at progressing and then at blocked replication forks.
In spite of the existence of a well-characterized multi-protein replication restart system, most often inactivated replication forks do not simply restart. A large panel of reactions can take place prior to restart, which depend on the mode of replication inactivation. Importantly, recombination proteins are involved in all these reactions; they act on their normal substrates, double-strand DNA breaks or single-strand DNA gaps present at blocked forks, and they also act directly on replication forks, catalyzing specific reactions. We will characterize the different steps involved in replication restart by setting up and improving an in vivo single-molecule microscopy technique called detection by localisation of the fluorescent signal. This technique is based on the possibility to detect a single fluorescent molecule if this molecule is immobilized for a long enough time (few msec.). For these experiments, we will construct a microscope with lasers with different wavelength to detect proteins labelled with different fluorophores, and then improve this technique by adding a stroboscopic illumination system, to increase the lifetime of the observed fluorophores, and a dual-view system to be able to observe two different fluorescent proteins at the same time.
We will label several replication and recombination proteins with a fluorescent tag. Using in vivo quantitative single molecule fluorescence, we will first analyze the kinetics of Okazaki fragments synthesis during replication progression. We will then directly measure the kinetics of replication protein disassembly after replication blockage and the kinetics of successive steps during fork remodeling and replication restart, including the action of different recombination proteins. Several genetically characterized conditions of replication blockage will be analyzed by single-molecule microscopy, allowing us to understand why these different types of replication blockage lead to different pathways of replication restart. This work will provide us with a unique opportunity to directly visualize the reactions that follow replication arrest and to decipher the rules that govern the reactivation of inactivated replication forks in a model system.