13 14 15 16 17 SUMMARY: 18 Faithful replication of chromatin domains during cell division is fundamental to eukaryotic 19 development. During replication, nucleosomes are disrupted ahead of the replication fork, followed 20by their rapid reassembly on daughter strands from the pool of recycled parental and newly 21 synthesized histones. Here, we use single-molecule imaging and replication assays in Xenopus laevis 22 egg extracts to determine the outcome of replication fork encounters with nucleosomes. Contrary to 23 current models, the majority of parental histones are evicted from the DNA, with histone recycling, 24 nucleosome sliding and replication fork stalling also occurring but at lower frequencies. The 25 anticipated local histone transfer only becomes dominant upon depletion of free histones from 26 extracts. Our studies provide the first direct evidence that parental histones remain in close proximity 27 to their original locus during recycling and reveal that provision of excess histones results in impaired 28 histone recycling, which has the potential to affect epigenetic memory. 29 30 (Word count: 148) 31 32 33 34 35 Eukaryotic genomes are organized into chromatin, which influences many cellular processes, ranging 36 from DNA replication and repair to gene transcription. The basic unit of chromatin is a nucleosome, 37 which consists of 145-147 base pairs of DNA wrapped around an octameric histone protein core, 38 formed from two copies of each of the four histones: H2A, H2B, H3 and H4. Histones H3 and H4 39 assemble into a symmetric hetero-tetramer and the two H2A-H2B dimers are docked onto the (H3-40 H4)2 tetramer (Luger et al., 1997). Nucleosomes are very stable nucleoprotein complexes but they are 41 also highly dynamic with regards to their conformation, composition and positioning within chromatin 42 (Lai and Pugh, 2017; Zhou et al., 2019). Nucleosome dynamics control DNA accessibility and are 43 regulated by complex interplay of numerous factors, such as chromatin remodelers, histone 44 chaperones, modifying enzymes and polymerases (Lai and Pugh, 2017).45 Chromatin is partitioned into domains, which either promote or block transcription, and 46 hence determine the cellular identity. Nucleosomes in transcriptionally active and silenced chromatin 47 domains carry specific histone post-translational modifications (PTMs) and/or distinct histone 48 sequence variants. The disordered tails of histones H3 and H4 are primary targets for PTMs associated 49 with different chromatin states; for example, tri-methylation of histone H3 at lysine 36 (H3-K36Me3) 50 and acetylation of histone H4 at lysine 16 (H4-K16Ac) mark transcriptionally active chromatin, whereas 51 tri-methylation of histone H3 at lysine 9 and 27 (H3-K9Me3 and H3-K27Me3) tag transcriptionally 52 silenced chromatin domains (Reinberg and Vales, 2018; Stillman, 2018). Therefore, maintenance of 53 cellular identity through mitotic cell division relies on faithful transfer of information encoded in both 54 DNA sequence (genetic inheritance) and n...