Rtt109 promotes nucleosome replacement ahead of the replicating fork

DNA replication perturbs chromatin by triggering the eviction, replacement and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a recently established sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing synchronous replication. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S phase-specific acetyltransferase, stabilizes nucleosomes behind the fork, but promotes H3 replacement ahead of the fork. Unexpectedly, increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56, and rather results from Rtt109 activity towards the H3 N-terminus. Our results suggest that selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, which may contribute to genome stability by overcoming replication-associated challenges.