Ruis K et al. (2022), Delineation of a minimal topoisomerase II bindi...

XB-ART-59237

J Biol Chem 2022 Jul 01;2987:101992. doi: 10.1016/j.jbc.2022.101992.

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Delineation of a minimal topoisomerase II binding protein 1 for regulated activation of ATR at DNA double-strand breaks.

Ruis K , Huynh O , Montales K , Barr NA , Michael WM .

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Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.

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Species referenced: Xenopus laevis
Genes referenced: atr brca1 chek1 lgals4.2 myc topbp1

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	Figure 1. Deletion analysis of TOPBP1 recruitment to DSBs. A, schematic summarizing the TOPBP1 proteins that were tested for binding to DSBs. The proteins are referred to by the letters A-E, at left, and also shown are the amino acid residues corresponding to each. B, schematic summarizing the DSB-binding assay. C, a representative experiment testing the ability of TOPBP1 deletion mutants to bind DSBs in XEE is shown. IVTT-expressed and myc-tagged proteins were mixed with XEE at 1 part IVTT lysate (5 Î¼l) to 4 parts XEE (20 Î¼l). DSB beads (600 fm of 5kb dsDNA in a volume of 5 Î¼l) were then added and the samples were incubated at room temperature for 30 min. The beads were then isolated back out of the extract, washed, and probed for occupancy of the indicated TOPBP1 deletion mutant. Panel â€œmyc boundâ€ refers to material that was bound to the DSB beads and the signal represents 20% of the total bound material. Panel â€œmyc inputâ€ refers to a sample of the total extract taken prior to addition of the DSB beads, and the signal represents 0.5% of the total amount present in the reaction. Panel â€œlow molecular weight protein boundâ€ is a silver-stained gel showing low molecular weight protein, likely histone, that bind DNA beads and not empty beads. This is used as a control for equal isolation of the DSB beads across the sample set. The experiment shown is representative of two independently performed replicates. DSBs, DNA double-strand breaks; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.
	Figure 2. Smaller, synthetic forms of TOPBP1 require the presence of XEE to bind DSBs. A, schematic showing the two synthetic forms of TOPBP1 that were used for DNA- and DSB-binding analysis. B, representative experiment testing the ability of the indicated TOPBP1 derivatives to bind dsDNA is shown. There is no XEE in this experiment. Twenty microliters of IVTT lysate expressing the indicated protein was incubated with 600 fmols of 5kb dsDNA immobilized on streptavidin beads (5 Î¼l volume of beads). After 30 min, the beads were isolated, washed, and probed for occupancy of the target protein by virtue of the myc epitope tag. â€œEmpty beadsâ€ refers to streptavidin beads lacking DNA. Panel â€œmyc boundâ€ refers to material that was bound to the DSB beads, and the signal represents 20% of the bound material. Panel â€œmyc inputâ€ refers to a sample of the total lysate taken prior to addition of the DSB beads, and the signal represents 0.5% of the total amount present in the reaction. The experiment shown is representative of two independently performed replicates. C, a representative experiment testing the ability of the indicated TOPBP1 derivatives to bind DSBs in the presence of XEE is shown. This was performed exactly as described in Figure 1C. Because the expression of full-length TOPBP1 was weaker than either Junior or III, we included a set of panels showing a darker exposure of the blot, so that the signals for full-length TOPBP1 are easier to see. The experiment shown is representative of three independently performed replicates comparing the binding of full-length TOPBP1 to Junior and two replicates comparing full-length TOPBP1 to Junior and III. AVG refers to average and SD refers to standard deviation. DSBs, DNA double-strand breaks; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.



	Figure 6. Summary and a model for Junior-mediated activation of ATR. A, a schematic summarizing how the different regions of TOPBP1 contribute to ATR activation at DSBs. B, a model for why Junior, but not III, can activate ATR. Please see text for details. ATR, ataxia telangiectasia and Rad3-related; DSBs, DNA double-strand breaks; TOPBP1, Topoisomerase II binding protein 1.
	Figure 1. Deletion analysis of TOPBP1 recruitment to DSBs.A, schematic summarizing the TOPBP1 proteins that were tested for binding to DSBs. The proteins are referred to by the letters A-E, at left, and also shown are the amino acid residues corresponding to each. B, schematic summarizing the DSB-binding assay. C, a representative experiment testing the ability of TOPBP1 deletion mutants to bind DSBs in XEE is shown. IVTT-expressed and myc-tagged proteins were mixed with XEE at 1 part IVTT lysate (5 μl) to 4 parts XEE (20 μl). DSB beads (600 fm of 5kb dsDNA in a volume of 5 μl) were then added and the samples were incubated at room temperature for 30 min. The beads were then isolated back out of the extract, washed, and probed for occupancy of the indicated TOPBP1 deletion mutant. Panel “myc bound” refers to material that was bound to the DSB beads and the signal represents 20% of the total bound material. Panel “myc input” refers to a sample of the total extract taken prior to addition of the DSB beads, and the signal represents 0.5% of the total amount present in the reaction. Panel “low molecular weight protein bound” is a silver-stained gel showing low molecular weight protein, likely histone, that bind DNA beads and not empty beads. This is used as a control for equal isolation of the DSB beads across the sample set. The experiment shown is representative of two independently performed replicates. DSBs, DNA double-strand breaks; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.
	Figure 2. Smaller, synthetic forms of TOPBP1 require the presence of XEE to bind DSBs.A, schematic showing the two synthetic forms of TOPBP1 that were used for DNA- and DSB-binding analysis. B, representative experiment testing the ability of the indicated TOPBP1 derivatives to bind dsDNA is shown. There is no XEE in this experiment. Twenty microliters of IVTT lysate expressing the indicated protein was incubated with 600 fmols of 5kb dsDNA immobilized on streptavidin beads (5 μl volume of beads). After 30 min, the beads were isolated, washed, and probed for occupancy of the target protein by virtue of the myc epitope tag. “Empty beads” refers to streptavidin beads lacking DNA. Panel “myc bound” refers to material that was bound to the DSB beads, and the signal represents 20% of the bound material. Panel “myc input” refers to a sample of the total lysate taken prior to addition of the DSB beads, and the signal represents 0.5% of the total amount present in the reaction. The experiment shown is representative of two independently performed replicates. C, a representative experiment testing the ability of the indicated TOPBP1 derivatives to bind DSBs in the presence of XEE is shown. This was performed exactly as described in Figure 1C. Because the expression of full-length TOPBP1 was weaker than either Junior or III, we included a set of panels showing a darker exposure of the blot, so that the signals for full-length TOPBP1 are easier to see. The experiment shown is representative of three independently performed replicates comparing the binding of full-length TOPBP1 to Junior and two replicates comparing full-length TOPBP1 to Junior and III. AVG refers to average and SD refers to standard deviation. DSBs, DNA double-strand breaks; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.
	Figure 3. A minimal TOPBP1 for regulated activation of ATR.A, schematic showing the synthetic forms of TOPBP1 that were used for DMAX assays. B, a representative experiment testing the ability of the indicated TOPBP1 derivatives to activate ATR. XEE was depleted of endogenous TOPBP1 and supplemented with either unprogrammed IVTT lysate (blank), or IVTT lysates programmed to produce myc-tagged forms of full-length TOPBP1, TOPBP1 Junior, or TOPBP1 III. For each sample, 20 μl of TOPBP1-depleted XEE was combined with 2.5 μl of IVTT lysate. The panel on the left shows the blank and full-length TOPBP1 samples probed with an antibody recognizing TOPBP1; this demonstrates that all detectable endogenous TOPBP1 was removed from the XEE. The panels on the right show the results of the DMAX assay. All samples received “lambda DSBs”, which is phage lambda DNA digested with the EcoRI restriction enzyme and added at a concentration of 20 ng/μl (see Experimental procedures). After a 30-min incubation, samples were recovered and probed by Western blotting for the indicated proteins. Shown below the blot is quantification of the P-CHK1 signal across multiple replicates. AVG stands for average and SD refers to standard deviation. The experiment shown is representative of three independently performed replicates comparing full-length TOPBP1 to Junior and two replicates comparing full-length TOPBP1 to Junior and III. C, a representative experiment testing the requirement for DSBs in TOPBP1 Junior-mediated activation of ATR. XEE (not depleted, 20 μl) was combined with 4 μl of the indicated IVTT lysate and then lambda DSBs were optionally added at 20 ng/μl. After a 30-min incubation, samples were recovered and probed by Western blotting for the indicated proteins. The experiment shown is representative of two independently performed replicates. D, a representative experiment examining the ability of the ΔBRCT4&5 mutant to activate ATR is shown. The experiment was performed exactly as described for Part B, above. The experiment shown is representative of two independently performed replicates. ATR, ataxia telangiectasia and Rad3-related; DSBs, DNA double-strand breaks; DMAX, DSB-mediated ATR activation in Xenopus; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.
	Figure 4. Replacement of BRCT0-2 with a heterologous DNA-binding domain allows ATR activation.A, schematic showing the forms of TOPBP1 that were used for DSB-binding and DMAX assays. B, schematic showing the 5kb dsDNA “DSB” used for DSB-binding and DMAX assays. Five copies of the UAS are present, positioned in the center of the molecule. C, a representative experiment testing the ability of IVTT-produced TOPBP1 Junior, or the GAL4 derivative, to bind the 5XUAS-containing DSB is shown. The experiment was performed exactly as in Figure 1C. For the Western blot, we probed the samples with an antibody raised against the BRCT7&8 domains of Xenopus TOPBP1 (see (20)). This antibody, termed HU142, thus recognizes the two IVTT-produced proteins as well as the endogenous TOPBP1. We note that the blot labeled “input” shows that all three proteins of interest were present at similar levels in the total extract. We show two different exposures of the DSB-bound samples because of the intensity disparity between the GAL4 signal and TOPBP1/TOPBP1 Junior signals. The experiment shown is representative of two independently performed replicates. D, a representative experiment comparing the ability of TOPBP1 Junior and the GAL4 derivative to activate ATR is shown. The experiment was performed exactly as in Figure 3B, with the exception that we also included a sample of undepleted XEE, so that the efficiency of ATR activation by the two test proteins could be compared to that promoted by endogenous TOPBP1. The experiment shown is representative of two independently performed replicates. E, a representative experiment asking if the GAL4 derivative still requires DSBs to activate ATR. The experiment was performed exactly as in Figure 3C. The experiment shown is representative of two independently performed replicates. DSBs, DNA double-strand breaks; ATR, ataxia telangiectasia and Rad3-related; BRCT, BRCA1 C-terminal repeat; DMAX, DSB-mediated ATR activation in Xenopus; IVTT, in vitro transcription and translation; TOPBP1, Topoisomerase II binding protein 1; UAS, upstream activator sequence; XEE, Xenopus egg extract.
	Figure 5. Addition of BRCT7&8 to the AAD allows for more efficient activation of ATR.A, schematic showing the different GST fusion proteins that were used for ATR activation assays. B, a representative experiment testing the indicated GST fusions for ATR activation is shown. The indicated GST fusions were added to XEE at the indicated concentrations. Incubation was carried out for the indicated time and then the samples were processed for Western blotting and probed for the indicated proteins. The lanes-labeled “PBS” refer to samples that received PBS instead of a GST fusion protein. The experiment shown is representative of two independently performed replicates. C, Western blots of sucrose gradient fractions. Each gradient was divided into nine fractions, with fraction #1 representing the top and fraction #9 the bottom of the gradients. Proteins sediment within the gradient based on their molecular mass, with the higher molecular mass proteins sedimenting at the bottom. In the blots, we see that GST-AAD-BRCT7&8 sediments at a lower position than does GST-AAD, indicative of a higher molecular mass. We also see the same pattern when TOPBP1 Junior and III are compared. The experiment shown is representative of two independently performed replicates. AAD, ATR activation domain; ATR, ataxia telangiectasia and Rad3-related; BRCT, BRCA1 C-terminal repeat; TOPBP1, Topoisomerase II binding protein 1; XEE, Xenopus egg extract.
	Figure 6. Summary and a model for Junior-mediated activation of ATR.A, a schematic summarizing how the different regions of TOPBP1 contribute to ATR activation at DSBs. B, a model for why Junior, but not III, can activate ATR. Please see text for details. ATR, ataxia telangiectasia and Rad3-related; DSBs, DNA double-strand breaks; TOPBP1, Topoisomerase II binding protein 1.

References [+] :

Bagge, Functions of TopBP1 in preserving genome integrity during mitosis. 2021, Pubmed