engleski

Chromatin dynamics and replication fork progression in mammals

My thesis research project has been carried out under the joint supervision of Dr. Geneviève Almouzni at the Curie Institute whose laboratory is affiliated to the Doctoral school CDV (complexité du vivant) from the University Paris 6 and Dr. Vlatka Zoldoš from the University of Zagreb. My primary interest has been to study chromatin replication in mammalian cells and factors that could influence this process during replication fork elongation. To introduce the topic, I will thus provide the basics concerning DNA replication to discuss our current knowledge concerning chromatin and its dynamics during replication. Then, I will describe the key histone chaperones identified to date and possibly involved in the process. I will give a particular emphasis on Asf1, anti silencing function 1, as an attractive candidate for the control of histone dynamics during replication given the recent discovery of a complex containing Asf1 and the proteins MCM2-7, known as the putative replicative helicase in mammals. This will enable me to present my experimental work, which consisted in the development of two complementary strategies to analyze chromatin replication. I will then provide results obtained using these approaches and how they could be exploited to gain insights into the importance of distinct chromatin parameters that can modulate specific aspects of replication fork progression. First, I have established a novel 3D analysis protocol for quantification of fork progression based on an in vitro replication elongation assay on individual nuclei derived from mammalian cell culture. This new assay and its application to the analysis of fork progression and its dependence on distinct sub-nuclear environments have formed the basis of a first manuscript. Second, I have developed the DNA molecular combing technique to assess changes in replication parameters when specific chromatin factors are targeted for alteration. Using this assay, I could find that Asf1 depletion in human cells leads to slower fork progression (elongation), loss of coordination of sister fork movement within a replicon, reduced fork recovery under conditions of replicative stress and a possible activation of additional origins of replication. These data reveal for the first time the impact of Asf1 depletion directly at the level of individual forks. I will discuss how absence of Asf1 causing unwinding defect during fork progression could in turn lead to a disruption of the predetermined spatiotemporal replication programme of a cell by affecting its basic unit, the replication bubble.

Chromatin; DNA replication; Fork progression; Histone chaperone; Asf1; Elongation assay; Molecular combing; Nuclear organization

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