Yan S , Michael WM .

GM 067735 NIGMS NIH HHS , R01 GM067735 NIGMS NIH HHS

	Figure 1. Requirements for the recruitment of TopBP1, pol-α, and 9-1-1 to stalled replication forks. (A) Experimental scheme for B–E. (B) NIB-250 chromatin was incubated in either mock- or TopBP1 (TopBP1−)-depleted extract that optionally contained aphidicolin (+Aph.). After a 45-min incubation, the chromatin-bound fraction was isolated using the ELB procedure. Checkpoint proteins on chromatin and Chk1 phosphorylation in total extract were further examined via Western blotting. (C) NIB-250 chromatin was added to TopBP1-depleted extract supplemented with either blank IVT (no add back) or recombinant IVT TopBP1 (+IVT TopBP1) and, optionally, aphidicolin. Checkpoint proteins on chromatin and Chk1 phosphorylation in extract were examined as in B. (D) Same as B except that pol-α, instead of TopBP1, was depleted in the second egg extract. (E) Either buffer (no add back) or 35 ng/µl purified DNA pol-α (+purified pol-α) was added to pol-α–depleted extract together with NIB-250 chromatin and, optionally, aphidicolin. Rad9 loading onto chromatin and Chk1 phosphorylation in extract were assessed as in B. (F) Experimental scheme for G. (G) Sperm chromatin was combined with mock- or Rad9 (Rad9−)-depleted extract and, where indicated, aphidicolin. Chromatin fractions and total extracts were analyzed as in B.
	Figure 2. Recruitment of pol-α and 9-1-1 to stalled replication forks is a novel function of TopBP1. (A) Summary of WT and mutant TopBP1s' functions in replication initiation, pol-α hyperloading, Rad9 loading, and Chk1 phospho-Ser344. A.D., activation domain; a, Yan et al., 2006; b, Kumagai et al., 2006. (B) IVT versions of TopBP1s were added back to TopBP1-depleled extract along with NIB-250 chromatin and aphidicolin. After incubation, the samples were processed for Western blotting as in Fig. 1 C. Additional Western blotting, using an antibody that recognizes the myc tag fused to all recombinant TopBP1s, confirmed that these recombinant forms of TopBP1s were added to extract in equivalent amounts (myc-TopBP1). (C) Blank IVT (−) or recombinant IVT TopBP1s (WT or W1138R) were added to TopBP1-depleted egg extract supplemented with sperm chromatin and aphidicolin. After incubation, the chromatin fractions and total extract were probed via immunoblotting. (D) Same as B except that WT or M5 TopBP1 was added back to TopBP1-depleled extract. (E) Blank IVT (−), WT TopBP1 (WT), or M3 TopBP1 (M3) was added back to TopBP1-depleted egg extract containing AT(70). The AT(70) DNA substrate used for this experiment was prepared as previously described (see Materials and methods; Yan et al., 2006). After incubation, Chk1 phosphorylation and myc-TopBP1 in total extract were determined via Western blotting.
	Figure 3. TopBP1 chromatin association is required for pol-α hyperloading and 9-1-1 recruitment to stalled replication forks. (A) Summary of WT and mutant TopBP1s' function in chromatin binding, pol-α hyperloading, Rad9 loading, and Chk1 phospho-Ser344. A.D., activation domain. (B) IVT forms of TopBP1s were added back to TopBP1-depleted egg extract combined with NIB-250 chromatin and, optionally, aphidicolin (+A). The chromatin fraction was isolated and examined for the presence of myc-TopBP1 via Western blotting. (C) Same as B except WT or W265R TopBP1 was added back. (D) Sperm chromatin was added to mock- or TopBP1-depleted (TopBP1 add back) extract optionally containing either blank IVT (−), WT, or W265R TopBP1. DNA synthesis was analyzed at 30 and 60 min as in Fig. S1 C. a.u., arbitrary unit. (E) Sperm chromatin was added to either mock- or TopBP1-depleted (TopBP1 add back) extract supplemented with blank IVT (−), WT, or W265R TopBP1 and, optionally, aphidicolin. Checkpoint proteins on chromatin and Chk1 phosphorylation in extract were assessed by immunoblotting.
	Figure 4. TopBP1 but not pol-α must be present with 9-1-1 for 9-1-1 to load onto stalled replication forks. (A) Experimental design for B and C. (B) NIB-250 chromatin isolated from aphidicolin-containing Rad9-depleted extract was transferred to a second either mock- or pol-α (pol-α−)–depleted extract that also contained aphidicolin. Checkpoint proteins on chromatin and Chk1 phosphorylation in extract were examined as in Fig. 1 B. (C) Same as B except that TopBP1 was depleted in the second extract.
	Figure 5. BRCT 1 and 2 of TopBP1 blocks the loading of 9-1-1 but neither TopBP1 nor pol-α onto stalled replication forks in a dominant-negative manner. (A) Either GST or GST–BRCT 1 and 2 was used for GST pull-down assays as described in Materials and methods. (B) Sperm chromatin was incubated in egg extracts supplemented with buffer, 926 nM GST, or 806 nM GST–BRCT 1 and 2. DNA synthesis products were analyzed at 30, 60, and 90 min as in Fig. S1 C. a.u., arbitrary unit. (C) Either 926 nM GST or 806 nM GST–BRCT 1 and 2 was preincubated with egg extracts, which were then supplemented with sperm chromatin and, optionally, aphidicolin (+Aph.). Checkpoint proteins on chromatin and Chk1 phosphorylation in extract were analyzed as in Fig. 1 B.
	Figure 6. A model for the roles of TopBP1 mediating 9-1-1 loading onto stalled replication forks. See Discussion for details.

Ball, ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation. 2005, Pubmed

Ball, ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation. 2005, Pubmed
Ball, Function of a conserved checkpoint recruitment domain in ATRIP proteins. 2007, Pubmed
Bermudez, Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. 2003, Pubmed
Burrows, How ATR turns on: TopBP1 goes on ATRIP with ATR. 2008, Pubmed
Byun, Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. 2005, Pubmed , Xenbase
Chen, Chk1 in the DNA damage response: conserved roles from yeasts to mammals. 2004, Pubmed
Cimprich, ATR: an essential regulator of genome integrity. 2008, Pubmed
Cortez, ATR and ATRIP: partners in checkpoint signaling. 2001, Pubmed
Delacroix, The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. 2007, Pubmed
Ellison, Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA. 2003, Pubmed
Errico, Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts. 2007, Pubmed , Xenbase
Furuya, Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1. 2004, Pubmed
Garcia, Identification and functional analysis of TopBP1 and its homologs. 2005, Pubmed
Guo, Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. 2000, Pubmed , Xenbase
Hashimoto, Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication. 2003, Pubmed , Xenbase
Hashimoto, The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1. 2006, Pubmed , Xenbase
Hubscher, Eukaryotic DNA polymerases. 2002, Pubmed
Jones, XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus. 2003, Pubmed , Xenbase
Kanoh, Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks. 2006, Pubmed
Kondo, Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms. 2001, Pubmed
Kumagai, TopBP1 activates the ATR-ATRIP complex. 2006, Pubmed , Xenbase
Lee, The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. 2007, Pubmed , Xenbase
Lee, Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. 2003, Pubmed , Xenbase
Lopes, Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. 2006, Pubmed
Luciani, Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus. 2004, Pubmed , Xenbase
Lupardus, A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. 2002, Pubmed , Xenbase
Lupardus, Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus. 2006, Pubmed , Xenbase
MacDougall, The structural determinants of checkpoint activation. 2007, Pubmed , Xenbase
Majka, Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions. 2006, Pubmed
Melo, Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. 2001, Pubmed
Michael, Activation of the DNA replication checkpoint through RNA synthesis by primase. 2000, Pubmed , Xenbase
Mimura, Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. 2000, Pubmed , Xenbase
Mordes, TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. 2008, Pubmed
Namiki, ATRIP associates with replication protein A-coated ssDNA through multiple interactions. 2006, Pubmed
Parrilla-Castellar, Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex. 2004, Pubmed
Parrilla-Castellar, Cut5 is required for the binding of Atr and DNA polymerase alpha to genotoxin-damaged chromatin. 2003, Pubmed , Xenbase
Paulsen, The ATR pathway: fine-tuning the fork. 2007, Pubmed
Rowles, Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. 1999, Pubmed , Xenbase
Stokes, DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts. 2002, Pubmed , Xenbase
Trenz, ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks. 2006, Pubmed , Xenbase
Unsal-Kaçmaz, Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities. 2004, Pubmed
Van Hatten, The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. 2002, Pubmed , Xenbase
Yan, Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling. 2006, Pubmed , Xenbase
You, The role of single-stranded DNA and polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint. 2002, Pubmed , Xenbase
Zhang, Turning the replication checkpoint on and off. 2006, Pubmed
Zhu, Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. 2007, Pubmed , Xenbase
Zou, Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. 2003, Pubmed
Zou, Replication protein A-mediated recruitment and activation of Rad17 complexes. 2003, Pubmed
Zou, Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. 2002, Pubmed