The eukaryotic replisome intrinsically generates asymmetric daughter chromatin fibers

DNA replication is molecularly asymmetric, due to distinct mechanisms for lagging and leading strand DNA synthesis. Whether chromatin assembly on newly replicated strands is also asymmetric remains unknown, as visualizing this short-lived state in cells is impossible. To circumvent this limitation, we combine in vitro reconstitution of the Saccharomyces cerevisiae DNA and chromatin replication machineries with replication-aware single-molecule chromatin footprinting, to study how chromatin is re-assembled on replicated DNA. Leveraging the non-destructive, single-molecule, and strand-specific nature of our data, we discover an intrinsic asymmetry in nucleosome positioning patterns and organization between lagging- and leading-strand chromatin created by the yeast replisome. This asymmetry is only partially restored upon addition of chromatin assembly factors involved in de novo histone deposition and the ATP-dependent chromatin remodeler Isw1a, implying that other regulatory factors must resolve this asymmetry in cells. In sum, our data reveal the complexity of chromatin re-establishment following DNA replication, and suggest an asymmetric chromatin assembly intermediate on each daughter chromatid. These pathways have implications for essential chromatin-templated processes such as DNA repair, transcription, and gene silencing at replication forks.