The structural determinants of checkpoint activation

Abstract

Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5'- and 3'-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage.

Figure 1.

Primed M13 ssDNA induces Chk1 phosphorylation and checkpoint activation. (A) M13 ssDNA (30 μg/mL) was added to NPE, a 1:1 mixture of cytosol and NPE, or cytosol (30 min) and then NPE. Parallel samples were removed at the indicated times post-DNA addition for NPE and cytosol + NPE. For the sequential addition, times were post-NPE addition. Samples were either immunoblotted for phospho-Chk1 (S344) and Chk1 (top panels), or replication was monitored by measuring α³²P-dCTP incorporation following gel electrophoresis (bottom). (B) Buffer, M13 ssDNA, the ssDNA primer (80-mer), or M13 preannealed to the primer (M13 + primer) was added to NPE. Samples were taken after 20 min and analyzed by immunoblotting after SDS-PAGE with antibodies for phospho-Chk1 (S344), Chk1, Rad1, and Cds1. (C) ssM13 DNA or M13 + primer was added to NPE at the indicated concentrations. Samples were analyzed as in B. (D) Double-stranded pBS (30 μg/mL) was incubated in cytosol for 30 min and added to an equal volume of NPE (lanes 1,2), NPE containing M13 + primer (20 min; 12.5 μg/mL; lanes 3,4), or NPE containing M13 + primer + caffeine (20 min; 12.5 μg/mL; caff, 4 mM; lanes 5,6). Parallel samples were removed 30 and 60 min post-addition of plasmid to NPE and analyzed for replication as in A (top panel) or immunoblotted for phospho-Chk1 (S344) and Chk1 (bottom panels). Final concentrations are shown.

Figure 2.

Effect of checkpoint protein depletion on phosphorylation events induced by primed M13 ssDNA. (A) Buffer, M13 + primer, or EcoRI-digested pBS (0.2 μg/mL) was added to mock-depleted or ATRIP-depleted NPE, and samples were analyzed as in Figure 1B. (B–E) NPE was mock-depleted or depleted with antibodies to RPA70 (B), TopBP1 (C), Rad1 (D), or Claspin (E). Mock-depleted NPE or depleted NPE was then incubated with M13 + primer, and samples were analyzed as in Figure 1B. Chk1 (B,C,E) and RPA (D) levels were monitored as loading controls. The lower band in E represents cross-reacting residual IgG in the extract from depletion.

Figure 3.

Effect of modifying primer ends on Chk1 and Rad1 phosphorylation induced by primed M13 ssDNA. (A) Names and figures of structures used in B–D. Dot represents the biotinylated end of the primer. (B) M13, M13 + unmodified 80-mer, or M13 + 80-mer blocked at the 3′-end with biotin was incubated with streptavidin and added to NPE. Samples were taken and analyzed for phospho-Chk1 (S344), Chk1, or Rad1 by immunoblotting, or for replication as described in Figure 1A. (C) M13 ssDNA annealed to an 80-mer blocked at the 3′-end, 5′-end, or 3′- and 5′-ends with biotin were incubated with streptavidin, added to NPE in the presence of aphidicolin, and analyzed as described in B. (D) M13 ssDNA annealed to an 80-mer blocked at the 5′-end with biotin was incubated with streptavidin, added to NPE in the absence or presence of aphidicolin, and analyzed as described in B.

Figure 4.

Effect of ssDNA gap size on Chk1 phosphorylation. (A) Names and figures of structures used in B. Dot represents the biotinylated end of the primer. (B) The structures shown were incubated with streptavidin and then added to NPE in the presence of aphidicolin. Samples were analyzed as in Figure 3B.