Montales K , Kim A , Ruis K , Michael WM .

GM122887 NIH HHS , R01 GM122887 NIGMS NIH HHS

             DNA repair

	Figure 1. Schematic of TOPBP1 highlighting the BRCT domains and the AAD. We produced two panels of mutants. Misfolding mutants are shown up top and the PBP mutants are shown at the bottom
	Figure 2. Linear dsDNAs trigger a legitimate DSB response upon incubation in HSS. (A) Experimental scheme. (B) The indicated amount of lambda DNA was incubated in 20 μl of HSS for 60 min. Samples were then probed by Western blot for P-CHK1 and CHK1. The sample labeled “water” did not receive any DNA. (C) 4 µg of EcoRI-digested lambda DNA (“DSB”) was optionally added to 20 μl of HSS and samples were processed as in (A). Samples were then probed for P-CHK1, CHK1, and P-ATM (P-Ser1981). (D) Either DMSO (lanes 1 and 2) or ATMi (KU55933, 50 µM, lane 3) was added to HSS, which was then optionally supplemented with EcoRI-digested lambda DNA (“DSB”). Samples were processed as in (A) and then probed for MRE11.
	Figure 3. Basic properties of the DMAX system. (A) DMAX assay where dsDNA fragments of the indicated size (in nucleotides) were added to HSS at the indicated concentration. After 60-min incubation the samples were probed for P-CHK1 and CHK1. (B) DMAX assay where dsDNA fragments of the indicated sizeconcentration were added to HSS at the indicated concentration. After 60-min incubation the samples were probed for P-CHK1 and CHK1. (C) DMAX assay where HSS and 150 fmol of a 5kB dsDNA fragment were incubated together and samples were taken at the indicated time points. ATRi was included in the final sample, at 100 μM. Samples analyzed as in (A). (D) HSS was immunodepleted using either non-specific IgG (sample “mock”) or HU142, and antibody that recognizes TOPBP1 (samples “TOPBP1- HSS + ”). The depleted extracts were then supplemented with either an unprogrammed IVTT reaction (sample “blank”) or IVTT reactions programmed for production of wild type TOPBP1 (“WT”) or the indicated TOPBP1 point mutant (“W1138R”). The reconstituted extracts were then supplemented with 150 fm of the 5 kB dsDNA fragment and, after incubation, the samples were probed by Western blotting for the indicated proteins. HU142, raised against Xenopus TOPBP144, was used to probe for TOPBP1.
	Figure 4. A DMAX-based DSB binding assay. (A) dsDNA (5kB) was coupled to magnetic streptavidin beads and then added to HSS at the indicated concentration of DNA. The sample labeled “–” received no beads. The sample labeled “empty beads” received only magnetic streptavidin beads. After a 60-min incubation samples were taken and probed for P-CHK1 and total CHK1. The dashed white line on the blots demarcates a lane from the original image that was removed because it is irrelevant. All samples shown were run on the same gel and blotted together on the same membrane at the same time. See Fig. S1 for the original scan. (B) dsDNA (5kB) was coupled to magnetic streptavidin beads and then added to HSS at the indicated concentration of DNA. One sample lacked DNA (“empty beads”). After a 60-min incubation the beads were isolated on a magnetic stand and the beads were then washed three times with PBS + 0.1% Triton X-100 and bound proteins were eluted using 2X Sample Buffer. Samples of the bound material, as well as the starting extracts (“input”) were then probed by Western blotting for TOPBP1. In addition, the bound samples were analyzed by silver staining, with a focus on a ~ 12.5 kDa low molecular weight (“l.m.w.”) showing that it is bound to the beads in a dose-dependent manner, as is TOPBP1.
	Figure 5. Mutational analysis of the roles of individual BRCT domains in recruitment of TOPBP1 to DSBs. (A) HSS was combined with IVTT-produced and myc-tagged TOPBP1 proteins. These proteins were the wild type form (“WT”) and the W to R BRCT domain misfolding mutants shown in Fig. 1. The numbers above the blot reflect the given BRCT domain containing the W to R mutation. DSB beads were then added and the samples were incubated for 60 min. After incubation, the beads were processed as in Fig. 3B and the samples were probed for the indicated protein. The myc antibody was used to probe for TOPBP1. The bound samples were also stained by Coomassie and the ~ 12.5 kDa l.m.w protein is shown. (B) Same as (A) except the Y to R BRCT misfolding mutants were analyzed. (C–E) Same as (A) except the indicated PBP mutant was analyzed. The l.m.w. protein was visualized by silver staining. For (E), the dashed white line on the blots demarcates a lane from the original image that was removed because it is irrelevant. All samples shown were run on the same gel and blotted together on the same membrane at the same time. See Fig. S1 for the original scan.
	Figure 6. Multiple TOPBP1 BRCT domains are required for ATR-directed phosphorylation of CHK1. A−E DMAX assays where HSS was depleted of endogenous TOPBP1 and then supplemented with the indicated IVTT-produced TOPBP1 mutants prior to the addition of DSBs. Mock refers to extract that was mock-depleted using non-specific IgG. Blank refers to a TOPBP1-depleted extract that received an unprogrammed IVTT reaction. After incubation, the samples were probed by Western blotting for P-CHK1, CHK1, and TOPBP1 (using anti-TOPBP1 antibody HU142)
	Figure 7. Addition of isolated BRCT domains in excess blocks ATR signaling. (A) Schematic representation of full-length TopBP1 and the recombinant GST-tagged TOPBP1 BRCT domains used in (B). (B) Overexpression assay where the indicated purified proteins were added to HSS at a 50-fold excess over endogenous TOPBP1 and preincubated for 15 min. DSBs were then added to the samples and incubated for 60 min. Samples were probed by Western blotting for P-CHK1 and CHK1. The sample in lane 1 did not receive DSBs. (C–E) Same as (B) except that indicated proteins were added to the assays at the indicated concentration. For (D), the dashed white line on the blots demarcates a lane from the original image that was removed because it is irrelevant. All samples shown were run on the same gel and blotted together on the same membrane at the same time. See Fig. S1 for the original scan.

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