Van C , Yan S , Michael WM , Waga S , Cimprich KA .

ES016486 NIEHS NIH HHS , R01 ES016486-10 NIEHS NIH HHS , R01 ES016486 NIEHS NIH HHS , R01 GM067735 NIGMS NIH HHS

	Figure 1. Small DNA products accumulate in response to aphidicolin. (A) A 9-kb plasmid was incubated in HSE and NPE containing α-[32P]dCTP with or without aphidicolin (15 µM Aph). Three samples were taken at the indicated times after NPE addition. DNA was isolated from the first sample, run on a denaturing polyacrylamide gel, and autoradiographed (top). The second was run on a chloroquine agarose gel and stained with SybrGold (middle; U-form refers to unwound DNA). The third was run on an SDS-PAGE gel and immunoblotted for Chk1 phosphorylation with PCNA as a loading control. (B) Lengths and intensities of DNA intermediates in A were calculated using ImageQuant software (n = 3). (C–E) Xenopus sperm chromatin (2,500 nuclei/µl) and α-[32P]dCTP were added to mock- or Pol-α–depleted LSE. Recombinant Pol-α complex was added where indicated. (C) Depletion was verified by immunoblotting with Orc2 as a loading control. (D) Aliquots taken at the indicated times were analyzed for replication. (E) Mock and depleted extracts were treated with 15 µM aphidicolin, and samples were analyzed for continued synthesis as in A.
	Figure 2. Primer synthesis and elongation continues at a stalled fork. (A and B) A 9-kb plasmid was replicated in HSE and NPE with 15 µM aphidicolin (Aph). Radionucleotides were added at the indicated times after NPE addition (A) or at the start of the reaction (B), and samples were analyzed as in Fig. 1 A. (C) Sperm chromatin (4,000 nuclei/µl) was replicated in HSE and NPE with 15 µM aphidicolin. 30 min after NPE addition, p27KIP was added, and 5 min later, α-[32P]dCTP was added. Samples were taken at the indicated times and analyzed for Chk1 phosphorylation and small nascent DNAs as in Fig. 1 A.
	Figure 3. The ATR–9-1-1 pathway is not required for primer synthesis. (A–D) Sperm chromatin (2,000 nuclei/µl) was replicated in mock- or ATRIP-depleted LSE with or without 15 µM aphidicolin (Aph). (A) Samples were immunoblotted to verify depletion. (B–D) Aliquots were taken at the indicated times and analyzed for replication (B), Chk1 phosphorylation (C), and primer synthesis (D) as in Fig 1. (E–G) Sperm chromatin (2,500 nuclei/µl) was replicated in mock- or Rad1-depleted LSE containing recombinant 9-1-1 complex and 15 µM aphidicolin as indicated. (E and G) Aliquots were analyzed for Chk1 phosphorylation (E) and primer synthesis (G) as in Fig. 1 A. (F) In parallel, chromatin was isolated from a third aliquot, and bound proteins were immunoblotted with the indicated antibodies.
	Figure 4. Relationship between nascent DNA synthesis, Pol-α hyperloading, and TopBP1 function. (A) Sperm chromatin (2,500 nuclei/µl) was replicated in LSE containing 30 µM aphidicolin (Aph). At the indicated times, parallel samples were analyzed for Chk1 phosphorylation and chromatin binding as in Fig. 3 (E and F). 5 min before each time point, an aliquot was incubated with α-[32P]dCTP and analyzed for nascent DNA synthesis as in Fig. 1 A. (B) Experimental schematic for C–F. Sperm chromatin (4,000 nuclei/µl) was replicated in LSE and isolated after 45 min to yield initiated chromatin. Initiated chromatin was replicated in mock- or TopBP1-depleted LSE containing α-[32P]dCTP and p27KIP with or without 30 µM aphidicolin. (C) Initiated chromatin was blotted for chromatin-bound proteins. (D) Replication was analyzed in mock- and TopBP1-depleted extracts lacking aphidicolin as in Fig. 1 D. (E) Parallel samples were taken 60 min after the addition of initiated chromatin and analyzed for Chk1 phosphorylation or chromatin binding as in Fig. 3 (E and F). (F) Primer synthesis was analyzed in aphidicolin-treated extracts as in Fig. 1 A, and intensities were calculated using ImageQuant software and normalized relative to the 90-min sample from mock-depleted extract. Error bars represent standard error (n = 8).
	Figure 5. Pol-δ, Pol-ε, and PCNA are required for the accumulation of small nascent DNAs. (A–F) Sperm chromatin (2,500 nuclei/µl) was added to mock-, Pol-δ–, Pol-ε–, or PCNA-depleted LSE with or without 15 µM aphidicolin (n = 3). (A and D) Depletion was verified by immunoblotting. (B and E) Replication was monitored in the absence of aphidicolin as in Fig. 1 D. (C) Synthesis of small nascent DNAs was monitored in aphidicolin-treated extracts and quantitated as in Fig. 1 B. (F) Small nascent DNAs were monitored as in C.
	Figure 6. Pol-δ, Pol-ε, and PCNA are required for optimal Chk1 phosphorylation. (A–C) Untreated or UV-irradiated (1,000 J/m2) sperm chromatin (2,500 nuclei/µl) was added to mock-, Pol-δ–, Pol-ε–, or PCNA-depleted LSE in the presence or absence of 15 µM aphidicolin (Aph). (A) Chk1 phosphorylation was analyzed as in Fig. 1 A. (B) Chk1 phosphorylation at 60 min was normalized to total Chk1 levels and quantitated using FluorChem analysis software. Error bars indicate standard error (n = 3). (C) Samples were taken at 30 min and analyzed for chromatin binding as in Fig. 3 F.
	Figure 7. Primer synthesis contributes strongly to Chk1 phosphorylation. (A) Schematic of purified ssM13 structures. Biotinylated ends are represented by a dot. (B) Purified ssM13 structures were coupled to streptavidin and added at 6 ng/µl to NPE containing 300 µM aphidicolin. Samples were taken after 20 min and blotted for the proteins indicated. Chk1 phosphorylation was quantitated using Photoshop. (C) Purified ssM13 and 6–80-mer structures were added to NPE containing 300 µM aphidicolin (aph). The indicated concentration of the 6–80 mer is shown, and ssM13 was added so the total concentration of DNA was 144 ng/µl. Sperm chromatin (2,500 nuclei/µl) was also replicated in HSE and NPE containing 15 µM aphidicolin. Samples were analyzed at 20 (structures) or 60 min (chromatin) for Chk1 phosphorylation and quantitated as in Fig. 6 B. (B and C) Error bars indicate standard error (n = 3).

Arias, Initiation of DNA replication in Xenopus egg extracts. 2004, Pubmed, Xenbase

Arias, Initiation of DNA replication in Xenopus egg extracts. 2004, Pubmed , Xenbase
Arias, Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. 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
Blow, Replication forks, chromatin loops and dormant replication origins. 2008, Pubmed
Bochman, The Mcm2-7 complex has in vitro helicase activity. 2008, Pubmed
Branzei, The DNA damage response during DNA replication. 2005, 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
Carty, Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts. 1996, Pubmed
Chang, Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities. 2006, Pubmed , Xenbase
Chang, DNA damage tolerance: when it's OK to make mistakes. 2009, Pubmed
Cimprich, Probing ATR activation with model DNA templates. 2007, Pubmed , Xenbase
Cimprich, ATR: an essential regulator of genome integrity. 2008, Pubmed
Delacroix, The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. 2007, Pubmed
DePamphilis, Replication of eukaryotic chromosomes: a close-up of the replication fork. 1980, Pubmed
Ellison, Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA. 2003, Pubmed
Friedberg, Suffering in silence: the tolerance of DNA damage. 2005, Pubmed
Friedel, ATR/Mec1: coordinating fork stability and repair. 2009, Pubmed
Fukui, Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts. 2004, Pubmed , Xenbase
Garner, Studying the DNA damage response using in vitro model systems. 2009, Pubmed , Xenbase
Hamdan, Motors, switches, and contacts in the replisome. 2009, Pubmed
Hashimoto, The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1. 2006, Pubmed , Xenbase
Heller, Replisome assembly and the direct restart of stalled replication forks. 2006, Pubmed
Hubscher, Eukaryotic DNA polymerases. 2002, Pubmed
Kochaniak, Proliferating cell nuclear antigen uses two distinct modes to move along DNA. 2009, Pubmed , Xenbase
Kumagai, TopBP1 activates the ATR-ATRIP complex. 2006, Pubmed , Xenbase
Kunkel, Dividing the workload at a eukaryotic replication fork. 2008, Pubmed
Lambert, Checkpoint responses to replication fork barriers. 2005, Pubmed
Langston, DNA replication: keep moving and don't mind the gap. 2006, Pubmed
Lee, The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. 2007, Pubmed , Xenbase
Liu, The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted. 2007, Pubmed
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, 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
Lupardus, Analyzing the ATR-mediated checkpoint using Xenopus egg extracts. 2007, Pubmed , Xenbase
Lupardus, A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. 2002, 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
McGarry, Geminin, an inhibitor of DNA replication, is degraded during mitosis. 1998, Pubmed , Xenbase
Méchali, DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. 1982, Pubmed , Xenbase
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
Murray, Cell cycle extracts. 1991, Pubmed
Navas, RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae. 1996, Pubmed
Nethanel, An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains. 1988, Pubmed
Nick McElhinny, Division of labor at the eukaryotic replication fork. 2008, Pubmed
Pacek, A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. 2004, Pubmed , Xenbase
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
Pursell, Yeast DNA polymerase epsilon participates in leading-strand DNA replication. 2007, Pubmed
Sasakawa, Accumulation of FFA-1, the Xenopus homolog of Werner helicase, and DNA polymerase delta on chromatin in response to replication fork arrest. 2006, Pubmed , Xenbase
Segurado, The S-phase checkpoint: targeting the replication fork. 2009, Pubmed
Shechter, Regulation of DNA replication by ATR: signaling in response to DNA intermediates. 2004, Pubmed , Xenbase
Shechter, ATR and ATM regulate the timing of DNA replication origin firing. 2004, Pubmed , Xenbase
Shiotani, Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks. 2009, Pubmed
Sogo, Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. 2002, Pubmed
Stadlbauer, DNA replication in vitro by recombinant DNA-polymerase-alpha-primase. 1994, Pubmed
Svoboda, Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation. 1995, Pubmed
Tourrière, Maintenance of fork integrity at damaged DNA and natural pause sites. 2007, Pubmed
Tutter, Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs. 2006, Pubmed , Xenbase
Waga, The DNA replication fork in eukaryotic cells. 1998, Pubmed
Waga, DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts. 2001, Pubmed , Xenbase
Walter, Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha. 2000, Pubmed , Xenbase
Walter, Regulated chromosomal DNA replication in the absence of a nucleus. 1998, Pubmed , Xenbase
Walter, Regulation of replicon size in Xenopus egg extracts. 1997, Pubmed , Xenbase
Yan, TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: implications for a replication restart-based mechanism for ATR checkpoint activation. 2009, Pubmed
Yan, Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling. 2006, Pubmed , Xenbase
Yan, TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks. 2009, Pubmed , Xenbase
Yanow, Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest. 2003, Pubmed , Xenbase
Yao, Replisome structure and conformational dynamics underlie fork progression past obstacles. 2009, Pubmed
Yoo, Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase. 2004, Pubmed , Xenbase
You, The role of single-stranded DNA and polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint. 2002, Pubmed , Xenbase
Zhu, Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. 2007, Pubmed , Xenbase
Zou, Replication protein A-mediated recruitment and activation of Rad17 complexes. 2003, Pubmed
Zou, Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response. 2007, Pubmed
Zou, Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. 2003, Pubmed