Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in vivo role of HAT1, we generated HAT1-deficient DT40 clone (HAT1(-/-)). HAT1(-/-) cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly. HAT1(-/-) cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.