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    • Transient transfection with ATR kinase

    2023-02-06

    Transient transfection with ATR kinase-dead (D2475→A) [30] and ATM kinase-dead (D2870→Ala and N2875→K) [31] constructs was performed using Fugene 6 (Roche Applied Science, Indianapolis). Three microliters of fugene 6+1μg plasmid was used in transfections using 6-well plates at 2×105 cells/well and assayed using flow cytometry 3 days later.
    Results
    Discussion We provide evidence that both Atm and Atr participate in gene silencing by maintaining heterochromatin on the Xi. Treatment of mouse fibroblast Carprofen with 2-AP caused histone acetylation to appear on the Xi and resulted in reactivation of a GFP reporter gene on the Xi. Individually interfering with the Atm or Atr function by two different methods, siRNA and the expression of dominant-interfering kinase-dead ATM and ATR genes, also caused GFP reactivation. Our failure to detect an increase in gene reactivation in response to ionizing radiation argues that the observed gene reactivation is not caused by DNA damage that accumulates when Atm or Atr is inhibited. Our findings extend the roles of Atm and Atr to the maintenance of gene silencing by heterochromatin and raise the possibility that developmental defects seen in A-T patients may be caused in part by chromatin abnormalities and aberrant gene expression. In view of the evidence for roles in the maintenance of heterochromatin, Atm and Atr may function in a heterochromatin checkpoint pathway that senses chromatin defects and helps restore normal heterochromatin structure. This may involve HDAC-2 which interacts with ATR in unirradiated cells [22] or the BASC complex [23] which includes ATM, ATR, HDAC1, and BRCA1 (which has been shown to control Xi heterochromatin [25]). Alternatively chromatin defects may induce ATM and ATR to interact with factors that restore heterochromatin.
    Acknowledgments
    Introduction The ATM and ATR protein kinases are key regulators of DNA damage signal transduction [1], [2]. ATM responds to double-strand breaks (DSBs), while ATR responds to almost all types of DNA damage, and also to stalling of replisomes. ATM and ATR are thought to be activated by interacting with sites of DNA damage, allowing them to phosphorylate multiple target proteins at Ser–Gln or Thr–Gln (S/T–Q) motifs, that often lie in clusters referred to as SCDs (S/T–Q cluster domains) [3], [4], [5]. Both kinases rapidly translocate to sites of DNA damage, by mechanisms that are not yet clear, and can directly phosphorylate other proteins associated with these sites, e.g. the core histone variant H2AX [6], [7], [8]. Although this can apparently occur without the help of accessory proteins [9], [10], phosphorylation of downstream targets of ATM and ATR requires other “mediator” proteins [11]. These include the BRCA1 breast and ovarian cancer susceptibility gene product, the MRN complex, MDC1/NFBD1 and 53BP1 [11]. 53BP1, originally identified in a two-hybrid screen with p53 [12], [13], is an important regulator of genome stability that protects cells against double-strand breaks [12], [13]. 53BP1 null mice are viable but are highly tumor-prone, have defects in IgG class switching and V(D)J recombination and are profoundly hyersenstive to IR probably due to a defect in non-homologous end-joining [14], [15], [16], [17], [18], [19]. Recent data indicate that 53BP1 is downregulated during the transition of precancerous stage to carcinomas [20], and even loss of a single 53BP1 allele in mice causes genome instability and lymphoma [18]. At the cellular level, 53BP1−/− mouse embryo fibroblasts (MEFs) are mildly hypersensitive to IR and show mild defects in the IR-induced G2 checkpoint [17]. Human cells depleted of 53BP1 using siRNA duplexes show a partial defect in the intra-S phase checkpoint and also show defects in IR-induced G2/M checkpoint after low doses of radiation [17], [21], [22]. CHK2 phosphorylation is delayed in 53BP1−/− deficient cells [17] and there is a marked decrease in the cross-reactivity of IR-treated cells with an antibody that recognises phospho-SQ/TQ motifs targeted by ATM/ATR [17]. Despite these observations, the precise molecular functions of 53BP1 that mediate its biological roles are not understood. It is generally assumed that whatever the molecular role of 53BP1, it is specific to DSBs (which activate ATM). This is largely based on the observation that although 53BP1 colocalises with ATM at DSBs, it does not translocate to sites of UV-induced DNA damage [23].