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Eukaryotic DNA replication: a single-molecule approach to the study of yeast replication on chromatin

Periodic Reporting for period 2 - REPLICHROMA (Eukaryotic DNA replication: a single-molecule approach to the study of yeast replication on chromatin)

Reporting period: 2020-03-01 to 2021-08-31

The object of study is the replication of DNA, which is a central process in all living organisms. It is carried out with high accuracy by a multi-protein complex called the replisome. But our understanding of the dynamics of this process, particularly in the context of compacted DNA (chromatin), is very incomplete.

Understanding DNA replication in its natural chromatin context is importance for society, because failure to copy DNA properly can lead to defects in the copied genome, slow down or stall the replisome and its DNA synthesis, and ultimately lead to genomic instability or cell death. The ability of certain cancer cells to handle genomic instability can drive intratumor heterogeneity and tumor evolution.

The overall objectives including examining the dynamics of the replisome on bare DNA and in the context of chromatin, at the single-molecule level. The single-molecule level is important because it facilitates the examination of the dynamics.
Biochemical preparation has been performed at different levels, for multiple proteins. The instrumentation for the single-molecule experimentation has been implemented and calibrated. The first results, on the dynamics of proteins involved in loading the first stage of the replisome, have been published.
The first results, on the dynamics of proteins involved in loading the first stage of the replisome, have been published. These results represent the current state of the art. Expansion of the project to include subsequent stages of replisome assembly, as well as chromatin, is in progress.
Scanning confocal images of two ORC molecules, labelled in green, bound to dsDNA (not fluorescent).