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Jan Breuer, David Emanuel Antunes Ferreira, Mike Kramer, Jonas Bollermann, Minou Nowrousian

• We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete \(\textit {Sordaria macrospora}\). ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of \(\textit {Neurospora crassa}\) that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in \(\Delta\)rtt109 and \(\Delta\)asf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of \(\it rtt109\) and \(\it asf1\) are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, \(\it chk2\) mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.