The CBP/p300 family of proteins comprises related acetyltransferases that coactivate signal-responsive transcription. with in the response to stalled DNA BAY 57-9352 replication. These observations suggest a broader role BAY 57-9352 for the p300/CBP acetyltransferases in the modulation of chromatin structure and function during DNA metabolic events as well as for transcription. The protein acetyltransferases p300 and CBP are transcriptional coactivators for signal transduction cascades that regulate cell cycle progression cellular growth differentiation and apoptosis. Studies in mice indicate that both p300 and CBP are required for normal development in a gene dose-dependent manner (37 60 indicating that p300 and CBP have at least some overlapping functions. In addition to forming a physical link between activated transcription factors and the basal transcriptional machinery p300/CBP can enhance signal-responsive transcription by acetylating chromatin-associated proteins and consequently modifying chromatin structure and function (16 26 In addition to transcriptional activity recent evidence suggests that p300/CBP may also interact directly with complexes that mediate chromatin metabolism. For example p300 has been shown to bind PCNA associate with newly synthesized DNA and stimulate DNA synthesis in vitro (30). In addition acetylation of Fen1 the endonuclease important for the removal of RNA primers during Okazaki fragment maturation by p300/CBP inhibits its DNA binding and nuclease activity (31). p300 also binds and acetylates DNA polymerase β which is involved in base excision repair (29). p300/CBP is also in a complex with and acetylates thymine DNA glycosylase the enzyme that recognizes and repairs mispaired BAY 57-9352 thymine and uracil groups (57). Further evidence that p300 plays a role during the response to DNA damage comes from the observation that acetylation of the RecQ helicase WRN by p300 facilitates the translocation of WRN protein from the nucleolus to nucleoplasmic foci (9). The WRN protein is critical for the resolution and restart of stalled DNA replication forks (47). Taken together these observations suggest that p300/CBP plays an important role during DNA synthesis following DNA damage or stalled replication. Chromosome replication is accomplished by initiating DNA replication forks at many origins along each chromosome. The cell cycle ensures that the new DNA strands are replicated only once per cell cycle by strictly regulating the temporal and spatial firing of these origins (36). The cell cycle checkpoints continuously monitor the genome to prevent the irreversible event of mitosis until DNA synthesis has been completed or until DNA aberrations have been resolved (28). In eukaryotic cells the DNA replication checkpoint which is thought to ensure that mitosis does not occur until the completion of S phase is dependent upon protein kinases that are related to the ATR/ATM family. ATR is a CD1E mammalian gene with homology to the gene mutated in the human genetic disease ataxia telangiectasia (ATM) (17 35 ATR and ATM share significant functional and sequence homology with (6) ESR1/MEC1 (50) and the (27) and (cells results in a defect in the DNA replication checkpoint. METHODS and MATERIALS Plasmids. The transgene is the 10.6-kb V5 epitope-tagged cDNA (41) fused to 5.2 kb of genomic DNA that is 5′ to the putative dCBP start site and cloned into the Yellow Carnegie 4 vector (provided by Pam Geyer). The pUAST-small interfering RNA (siRNA) vector was generated by inserting BAY 57-9352 intron 7 of dCBP between inverted segments of coding sequence. The PCR fragment from bp 231 to 1139 was tagged with EcoRI and NotI the PCR fragment corresponding to the seventh intron was tagged with NotI and XhoI and the inverse 231- to 1131-bp PCR fragment was tagged with XhoI and XbaI. These fragments were cloned sequentially into the pUAST transformation vector (10). The pPacB expression construct was generated by cloning two PCR fragments from cDNA into pPacB (Invitrogen). The PCR fragment from bp 1 to 4415 was generated using the oligonucleotides 5′ NotI-AACAGCGGCCGCATGTCGACACAACGGAAGGAT and 3′ PCR fragment was generated with the oligonucleotides 5′ Kc cells were grown at 25°C in 1× Schneider’s medium (GIBCO) and supplemented with 5% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Kc cell transfections were performed using a BAY 57-9352 calcium phosphate transfection kit from Invitrogen according to the manufacturer’s protocol. Fly culture and strains. {The UAS-dCBP-siRNA and PThe Pyt7 and UAS-dCBP-siRNA.7 dCBPt + 16transgenic lines were.