Yan S and Michael WM (2009), TopBP1 and DNA polymerase-alpha directly recrui...

XB-ART-39370

J Cell Biol 2009 Mar 23;1846:793-804. doi: 10.1083/jcb.200810185.

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TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks.

Yan S , Michael WM .

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TopBP1 and the Rad9-Rad1-Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR-ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-alpha to sites of replication stress. Furthermore, we show that pol-alpha is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-alpha, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

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Species referenced: Xenopus laevis
Genes referenced: atr atrip chek1 ercc4 hus1 myc rad1 tbx2 topbp1

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	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.

References [+] :

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