Rad53 is required for degradation of excess histone protein not packaged into chromatin ( Gunjan and Verreault, 2003) and plays a role in histone metabolism and chromatin assembly ( Emili et al., 2001 Sharp et al., 2005). Checkpoints are also involved in histone and S phase coordination. DNA replication failure blocks further initiation from later-firing replication origins ( Painter and Young, 1980), stabilizes arrested replisome components arising from previously fired origins ( Dimitrova and Gilbert, 2000 Feijoo et al., 2001) and also blocks entry into mitosis ( Rao and Johnson, 1970). Replication stress induces phosphorylation of UPF1 but not hairpin-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase–like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK–mediated phosphorylation of UPF1.Ĭheckpoints control cell cycle timing after genomic insult ( Zhou and Elledge, 2000).
Histone mRNA decay does not require Chk1/Chk2. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress–induced histone mRNA down-regulation. Here, we expose S phase–arrested cells to the kinase inhibitors caffeine and LY294002. DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression.
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