A Piece-by-piece Replica of Chromatin: Building-Up and Dissecting Chromatin Dynamics During DNA Replication

The genome is packaged into chromatin, a dynamic structure essential for DNA organization and gene regulation. Fundamental cellular processes, such as DNA replication, pose a challenge to the maintenance of cell identity. During the S phase of the cell cycle, chromatin is disrupted to enable genome duplication. Simultaneously, chromatin is reassembled to preserve epigenetic information. These complex mechanisms are coordinated by individual factors that operate across multiple pathways. However, the molecular basis of epigenetic inheritance remains poorly defined. During DNA replication, recycled parental histones provide only half of the epigenetic material required on newly synthesized DNA, while the remainder must be generated de novo. Previous studies primarily focused on the restoration of epigenetic marks, such as histone modifications, through recycling mechanisms. However, how recycled and newly synthesized components are coordinated to maintain chromatin organization remains largely unclear. This thesis investigates the molecular principles governing the interplay between histone recycling and de novo chromatin assembly during DNA replication. Using a bottom-up biochemical reconstitution approach, we sequentially integrated purified replication and chromatin assembly factors to recapitulate complete pathways in vitro. In parallel, a genomics approach was developed to characterize the strand-specific organization of replicated chromatin at the nucleosome level. We uncovered an inherent asymmetric state in chromatin fiber organization between the two sister chromatids. This ground state potentially reflects fundamental asymmetries in DNA synthesis and histone recycling on each strand, before chromatin maturation and other downstream processes take place in cells. Furthermore, we mechanistically characterize the de novo chromatin assembly machinery, Chromatin Assembly Factor 1 (CAF-1). We demonstrate that CAF-1 uses independent, asymmetric mechanisms to assemble nucleosomes on each daughter strand and that chromatin assembly immediately behind the replication fork actively modulates DNA replication speed. Together, these findings provide the first characterization of nascent, strand-specific chromatin architecture and motivate further investigation into how these initial asymmetries are ultimately resolved to produce identical sister chromatids.