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 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 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