Replication independent ATR signalling leads to G2/M arrest requiring Nbs1, 53BP1 and MDC1

Abstract

Ataxia telangiectasia and Rad3-related (ATR) is a phosphoinositol-3-kinase like kinase (PIKK) that initiates a signal transduction response to replication fork stalling. Defects in ATR signalling have been reported in several disorders characterized by microcephaly and growth delay. Here, we gain insight into factors influencing the ATR signalling pathway and consider how they can be exploited for diagnostic purposes. Activation of ATR at stalled replication forks leads to intra-S and G2/M phase checkpoint arrest. ATR also phosphorylates gamma-H2AX at single-stranded (ss) DNA regions generated during nucleotide excision repair (NER) in non-replicating cells, but the critical analysis of any functional consequence has not been reported. Here, we show that UV irradiation of G2 phase cells causes ATR-dependent but replication-independent G2/M checkpoint arrest. This process requires the Nbs1 N-terminus encompassing the FHA and BRCT domains but not the Nbs1 C-terminus in contrast to ATM-dependent activation of G2/M arrest in response to ionizing radiation. Thus, Nbs1 has a function in ATR signalling in a manner distinct to any role at stalled replication forks. Replication-independent ATR signalling also requires the mediator proteins, 53BP1 and MDC1, providing direct evidence for their role in ATR signalling, but not H2AX. Finally, the process is activated in Cockayne's syndrome but not Xeroderma pigmentosum group A cells providing evidence that ssDNA regions generated during NER are the ATR-pathway-specific activating lesion. Replication-independent G2/M checkpoint arrest represents a suitable assay to specifically identify patients with defective ATR signalling, including Seckel syndrome, Nijmegen breakage syndrome and MCPH-1-dependent primary microcephaly.

Figure 1.

(A) G2/M arrest 1.5 h after UV in the presence of aphidicolin (APH) is dependent on ATR and XP-A. Control (WT), ATR-Seckel syndrome (ATR), A-T (AT) or XP-A (XP-A) LBLs were untreated or treated with 5 Jm⁻², incubated for 1.5 h with APH and the percent mitotic cells (MI, mitotic index) estimated. (B) Nbs is required for UV-induced replication-independent G2/M arrest. Cells were untreated or irradiated as in (A) but with 3 Jm⁻² UV. Cells with defective Nbs1 (nbs) fail to activate G2/M arrest. NBS cells corrected with full-length Nbs1 (nbs+p95) displayed normal G2/M arrest. (C) 53BP1 and MDC1 but not H2AX are required for UV-induced replication-independent G2/M arrest. Control (MDC1^+/+), MDC1 null (MDC1^−/−), 53BP1 null (53BP1^−/−) or H2AX null (H2AX^−/−) MEFs were untreated or treated as in (A).

Figure 2.

(A) Nbs constructs used. (B) Expression of Nbs1 and Mre11 by IF. (C) The N-terminus of Nbs is required for replication-independent G2/M arrest. Cells were untreated or irradiated with 3 Jm⁻², incubated for 1.5 h in the presence of APH and the MI counted. Cells lacking the Nbs1 N-terminus (nbs+FR5) fail to activate G2/M arrest. NBS cells corrected with an Nbs1 fragment lacking the C-terminal (nbs+652), or lacking only the ATM interacting region (nbs+dATM), or Nbs1 mutated in two phosphorylation sites (nbs+PP) displayed normal G2/M arrest. Results with Nbs and Nbs+p95 cells are shown in Fig. 1B. (D) The Nbs1 N-terminus is dispensable for G2/M checkpoint arrest following IR. Cells were untreated or irradiated with the indicated dose of IR, incubated for 1.5 h in the presence of APH and% mitotic cells (MI) counted.

Figure 3.

(A) The N-terminus of Nbs is required for the G2/M checkpoint after UV. The cells indicated were untreated or irradiated with 3 Jm⁻², incubated for 5 h in the presence of Nocodazole (no APH) and the MI counted. (B) Chk1 phosphorylation is dependent on the Nbs1 N-terminus after UV or HU treatment. Cells were treated with HU (1 mm) or UV (5 Jm⁻²) and 2 h later samples prepared for western blotting using α-p-Chk1 antibodies. (C) Quantification of (B). Western blots were performed as in (B) and at least three blots quantified by ImageQuant software. (D) The N-terminal of Nbs is required for recovery from replication stalling. Replication forks were pulse labelled with CldU and then arrested by treating with APH. Prior to removal of APH, cells were treated with IdU so that fork progression after replication stalling can be visualized. After removal of APH, cells recovered for 10 min. Restart represented the overlap of CldU (red) and IdU (green) labelling.

Figure 4.

XP-A but not CS-A or CS-B cells are defective in replication- independent G2/M arrest. Control (WT), XP-A, Cockayne syndrome A or B (CS-A, CS-B) transformed fibroblasts were untreated or treated with 5 Jm⁻², incubated for 1.5 h and the% mitotic cells estimated.