Mechanisms of ATM/ATR function and regulation.

摘要

ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are structurally and functionally related protein kinases belonging to the PIKK gene superfamily. ATM/ATR have been implicated as critical regulators of cellular responses to genotoxic stress including cell cycle checkpoint activation, DNA repair, gene transcription, and induction of apoptosis. Although many of the signaling pathways that are regulated by ATM/ATR are well understood, the manner in which these kinases are activated is not. We identified replication protein A (RPA), a single-stranded DNA-binding protein complex critical for DNA repair, replication, and recombination, as a factor required for targeting of ATR to damaged DNA. RPA-deficient cells exhibited delayed S phase progression, G2/M cell cycle arrest, apoptosis, high levels of spontaneous DNA damage, and constitutive ATM activation. Surprisingly, RPA-deficiency did not ablate replication stress-induced phosphorylation of the canonical ATR substrate CHK1. These findings reveal that RPA is required for genomic stability and that ATR activation can occur through RPA-independent mechanisms. We then characterized the mechanism of cyclic AMP responsive element-binding protein, CREB phosphorylation in response to replication stress. The kinetics of CREB phosphorylation were slow in comparison to CHK1, but correlated with ATM activation. Utilizing siRNA, we observed UV damage-induced phosphorylation of CREB exhibited ATR and ATM dependence. Moreover, UV light-induced CREB phosphorylation was absent in ATM-deficient cells. ATR RNAi caused reduction in UV light-stimulated ATM activation, suggesting that ATM functions downstream of ATR following replication stress. Since the UV light-induced activation of ATM by ATR appeared indirect we sought to determine other factors downstream of ATR in this process. Using a genetic approach, BLM helicase was identified as another protein required for UV light-induced ATM activation. Following replication stress, BLM is phosphorylated in an ATR-dependent manner. BLM-deficient cells displayed a defect in ATM activation in response to UV light. This defect is rescued in BLM-deficient cells complemented with wild-type BLM, but not in cells complemented with a phosphorylation-resistant mutant. These findings suggest a linear pathway by which ATR and BLM cooperate to activate ATM following replication stress.