Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage. Author Summary Loss of replication fork integrity is a primary source of genetic instability. In eukaryotes DNA synthesis is rapidly followed by its assembly into chromatin, and these two processes are tightly connected. Defective chromatin assembly mutants accumulate DNA damage and are sensitive to genotoxic agents, even though the mechanisms responsible for this genetic instability remain unclear because chromatin assembly also plays essential roles in transcription, silencing, DNA repair, and checkpoint signaling. A good example is the acetylation of histone H3 at lysine 56, which promotes histone deposition by the chromatin assembly factors CAF1 and Rtt106. In this case, the absence of this modification also causes a loss of structural and/or coding information at chromatin. Here we show that defective replication-coupled chromatin assembly leads to an accumulation of recombinogenic DNA damage by affecting the integrity of advancing, but not stalled, replication forks. Therefore, we propose that H3K56ac/CAF1/Rtt106-dependent chromatin assembly provides a mechanism for the stabilization of replication forks. Besides, H3K56 acetylation promotes replicative DNA damage repair/tolerance through a function that is independent of CAF1/Rtt106 and likely subsequent to its deposition at chromatin, revealing this modification as a key regulator of genome integrity.
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