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DNA sequence and epigenetic information embedded in chromatin must be faithfully duplicated and transmitted to daughter cells during cell division. However, how chromatin assembly and DNA replication are integrated remains unclear. We examined the contribution of the Tousled-like kinases 1 and 2 (TLK1/TLK2) to chromatin assembly and maintenance of replication fork integrity. We show that TLK activity is required for DNA replication and replication-coupled nucleosome assembly and that lack of TLK activity leads to replication fork stalling and the accumulation of single-stranded DNA, a phenotype distinct from ASF1 depletion. Consistent with these results, sustained TLK depletion gives rise to replication-dependent DNA damage and p53-dependent cell cycle arrest in G1. We find that deficient replication-coupled de novo nucleosome assembly renders replication forks unstable and highly dependent on the ATR and CHK1 checkpoint kinases, as well as poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) activity, to avoid collapse. Human cancer data revealed frequent up-regulation of TLK genes and an association with poor patient outcome in multiple types of cancer, and depletion of TLK activity leads to increased replication stress and DNA damage in a panel of cancer cells. Our results reveal a critical role for TLKs in chromatin replication and suppression of replication stress and identify a synergistic lethal relationship with checkpoint signaling and PARP that could be exploited in treatment of a broad range of cancers.

DNA sequence and epigenetic information embedded in chromatin must be faithfully duplicated and transmitted to daughter cells during cell division. However, how chromatin assembly and DNA replication are integrated remains unclear. We examined the contribution of the Tousled-like kinases 1 and 2 (TLK1/TLK2) to chromatin assembly and maintenance of replication fork integrity. We show that TLK activity is required for DNA replication and replication-coupled nucleosome assembly and that lack of TLK activity leads to replication fork stalling and the accumulation of single-stranded DNA, a phenotype distinct from ASF1 depletion. Consistent with these results, sustained TLK depletion gives rise to replication-dependent DNA damage and p53-dependent cell cycle arrest in G1. We find that deficient replication-coupled de novo nucleosome assembly renders replication forks unstable and highly dependent on the ATR and CHK1 checkpoint kinases, as well as poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) activity, to avoid collapse. Human cancer data revealed frequent up-regulation of TLK genes and an association with poor patient outcome in multiple types of cancer, and depletion of TLK activity leads to increased replication stress and DNA damage in a panel of cancer cells. Our results reveal a critical role for TLKs in chromatin replication and suppression of replication stress and identify a synergistic lethal relationship with checkpoint signaling and PARP that could be exploited in treatment of a broad range of cancers.

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.