Figure 4: Chk1 binds to PAR via the PbR motif and facilitates its kinase activity. | Nature Communications

Figure 4: Chk1 binds to PAR via the PbR motif and facilitates its kinase activity.

From: Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Figure 4

(a) Dot-blot assay of PAR binding of Chk1. Indicated protein (1 pmol) was spotted onto a nitrocellulose membrane and incubated with 32P-labelled PAR. The blots were washed with TBS-T buffer containing 600 mM NaCl plus 20 mM EDTA (lower panels). PAR-bound proteins were then visualized by autoradiography. Positive controls (Histone H1 and H4) and negative controls (BSA and GST) were included. Loading of GST-tagged proteins was confirmed by staining with an anti-GST antibody. (b) Identification of the putative PAR-binding motif in Chk1. A putative PAR-binding motif in Chk1 and was designated the ‘PAR-binding regulatory (PbR) motif’. Note that the PbR site (indicated in red) is highly conserved among vertebrates but not in yeast. (c) Chk1 kinase activity assay. GST-tagged wild-type Chk1 (GST-wtChk1) or PbR-mutant Chk1 (GST-PbRC/AChk1) proteins were incubated with 2 μg of GST-Cdc25a and [γ-32P]-ATP. 32P incorporation was detected by autoradiography. GST-tagged protein was detected by an anti-GST antibody. The Chk1 inhibitor UCN-01 was added to 100 ng GST-Chk1 as a control. (d) The kinase activity is measured by 32P incorporation in Chk1 or Cdc25a normalized for signal intensity of 100 ng wtChk1 input (GST signal) (c). (e) The kinase reaction was performed using recombinant GST-wtChk1 or GST-PbRC/AChk1 (100 ng). These proteins were incubated with size-fractionated PAR (S: 26–30 units; L: >65 units; S+L: 1:1 mixture of short- and long-chain PAR). PAR:GST-Chk1: molar ratio of (ADP-ribose) units within the PAR polymer to GST-Chk1 molecules, 10 or 40, respectively. The amount of ADP ribose units and thus negative charges are similar in the reactions with either long or short PAR polymer. 32P incorporation was detected by autoradiography. GST-tagged protein was detected by an anti-GST antibody. Phospho-Chk1 was blotted by a specific antibody against p-Chk1(S317). The Chk1 inhibitor UCN-01 was used as a control. The asterisk indicates an unspecific band. (f) Quantification of 32P incorporation in Chk1 is shown in panel e.

Back to article page