Yan S et al. (2006), Direct requirement for Xmus101 in ATR-mediated ...

XB-ART-439

J Cell Biol 2006 Apr 24;1732:181-6. doi: 10.1083/jcb.200601076.

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Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.

Yan S , Lindsay HD , Michael WM .

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TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3-related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

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Species referenced: Xenopus laevis
Genes referenced: antxr1 atr chek1 clspn mcm5 rrad tbx2 topbp1

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	Figure 1. Xmus101 is required, but replication proteins are not, for Chk1 activation in the AT70 system. (A) Recombinant Chk1ΔKD was added to extract, along with no DNA (lane 1), the A70 oligonucleotide (lane 2), or the preannealed AT70 oligonucleotide (lane 3). After a 100-min incubation, samples were taken and the phosphorylation status of Chk1ΔKD was assessed by SDS-PAGE and immunoblotting with the T7 monoclonal antibody (Novagen), which recognizes the T7 epitope tag present on recombinant Chk1ΔKD. The retarded mobility of Chk1ΔKD in lane 3 is due to phosphorylation (Michael et al., 2000). (B) MCM5 was immunodepleted from egg extract. The depleted extract was probed for MCM5 by immunoblotting and compared with a mock-depleted extract. (C) Same as B except that the p70 subunit of pol α was immunodepleted. (D) Mock-depleted and depleted extracts were supplemented with recombinant Chk1ΔKD and AT70. After a 100-min incubation, samples were taken and processed as in A. The lanes labeled “no DNA” refer to extracts incubated without AT70 but with recombinant Chk1ΔKD. (E) Xmus101 was immunodepleted from egg extracts. The depleted extracts were probed for Xmus101 by immunoblotting and compared with a mock-depleted extract. Add-back refers to Xmus101-depleted extracts supplemented with recombinant Xmus101. (F) The extracts depicted in E were assayed for Chk1ΔKD phosphorylation as in A.
	Figure 2. Two inhibitors of the Xmus101 Chk1 activation function. (A) A cartoon of Xmus101 showing the region of the protein that was used as antigen to produce the HU142 antibody as well as recombinant proteins used in this study. The numbered boxes refer to BRCT domains (Garcia et al., 2005). (B) Extracts were supplemented with either nonspecific IgG (lane 1) or HU142 (lane 2; final concentration: 50 ng/μl). Both samples were then further supplemented with recombinant Chk1ΔKD and AT70. After a 100-min incubation, samples were taken and probed for Chk1ΔKD as in Fig. 1 A. (C) Either GST (15 μM; lane 1) or GST-CT333 (806 nM, lane 2; 322 nM, lane 3; 161 nM, lane 4) was added to egg extract along with AT70 and Chk1ΔKD. After a 100-min incubation, samples were taken and probed for Chk1ΔKD as in Fig. 1 A.
	Figure 3. Xmus101 functions after assembly of the Claspin–Chk1 complex during AT70-mediated Chk1 activation. (A) A depiction of the Chk1 activation pathway. (B) Extracts were supplemented with recombinant GST-Claspin CKBD and the following additional components: buffer (lane 1), the A70 oligonucleotide (lane 2), preannealed AT70 oligonucleotides (lane 3), or preannealed AT70 oligonucleotides plus HU142 (final concentration: 50 ng/μl; lane 4). GST-Claspin CKBD phosphorylation was assessed after a 100-min incubation, according to published procedures (Kumagai and Dunphy, 2003). (C) GST-Claspin CKBD shift assay in extracts containing AT70 and buffer (lane 1), GST (15 μM; lane 2), or GST-CT333 (806 nM; lane 3). (D) Extracts were supplemented with bead bound recombinant Xchk1-GH (Kumagai and Dunphy, 2000) and the following additional components: buffer (lane 1), the A70 oligonucleotide (lane 2), preannealed AT70 oligonucleotides (lane 3), or preannealed AT70 oligonucleotides plus HU142 (final concentration: 50 ng/μl; lane 4). After a 100-min incubation, the beads were isolated and washed, and bound proteins were eluted with 2× SDS-PAGE sample buffer. The presence of Claspin and Xchk1-GH in the eluate was then determined by immunoblotting with antibodies against Claspin and GST, respectively (Jeong et al., 2003). (E) Claspin-Chk1 binding assay in extracts containing AT70 and buffer (lane 1), GST (15 μM; lane 2), or GST-CT333 (806 nM; lane 3).
	Figure 4. A separation-of-function mutant of Xmus101. (A) Schematic representations of full-length Xmus101 and Mini. (B) Constructs encoding either full-length Xmus101 (FL) or Mini were transcribed and translated in vitro in rabbit reticulocyte lysates in the presence of [35S]methionine. The radio-labeled proteins were then run out on SDS-PAGE and visualized after autoradiography. (C) Xmus101 was depleted from egg extract. Depleted extract was then supplemented (0.1 vol) with either unprogrammed reticulocyte lysates (Xmus101−) or lysates expressing either FL or Mini Xmus101. A mock-depleted sample was also prepared. The reconstituted extracts were then combined with sperm chromatin and α-[32P]dATP, and DNA replication was measured according to standard procedures (Walter and Newport, 1999). Samples were taken at 30, 60, and 90 min for analysis. (D) The reconstituted extracts described in C were supplemented with sperm chromatin and the DNA replication inhibitor aphidicolin (100 μg/ml). After a 60-min incubation, the samples were probed by immunoblotting for both activated and total Chk1.
	Figure 5. HU142 allows mitosis in aphidicolin-treated cycling extracts. (A) Cycling extracts were prepared and supplemented with sperm chromatin (1,000/μl). Extracts were further supplemented, where indicated, with aphidicolin, HU142, or 5 mM of caffeine. Entry into mitosis was determined by nuclear envelope breakdown. (B) Egg extracts were prepared and supplemented with sperm chromatin and HU142, as indicated. DNA replication was then assessed as in Fig. 4 C.

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