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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: DNA Repair (Amst). 2020 Sep 21;96:102973. doi: 10.1016/j.dnarep.2020.102973

Biochemical analysis of TOPBP1 oligomerization

Ahhyun Kim 1,2,6, Katrina Montales 1,6, Kenna Ruis 1,6, Holly Senebandith 1,3,6, Hovik Gasparyan 1,4, Quinn Cowan 1,5, W Matthew Michael 1,7
PMCID: PMC7670859  NIHMSID: NIHMS1631045  PMID: 32987353

Abstract

TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1’s BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.

Keywords: ATR, checkpoint, BRCT, DNA damage, scaffold

1. Introduction

Scaffold proteins play important roles in a diverse array of cellular signaling events. Scaffolds function by bringing together the different components of signal transduction pathways, thereby nucleating signaling centers within the cell. One scaffold protein that is crucial to the DNA damage checkpoint is TOPBP1. TOPBP1 is a large 180 kDa protein that is characterized by the presence of 9 BRCA1 C-terminus (BRCT) domains, dubbed BRCT0–8, that are scattered along the length of the protein (Koonin et al., 1996, Wardlaw et al., 2014). BRCT domains are interaction modules that bind a variety of substrates, including phosphorylated and unphosphorylated proteins, PARP chains, ubiquitin moieties, and DNA (Gerloff et al., 2012, Reinhardt and Yaffe, 2013, Wan et al., 2016). The presence of 9 BRCT domains within TOPBP1 suggests the ability to simultaneously interact with multiple factors during the DNA damage response.

One major function of TOPBP1 is to activate the ATR protein kinase at sites of DNA replication stress, or DNA double-strand breaks (DSBs; Saldivar et al., 2017). TOPBP1 is recruited to sites of damage via an unknown mechanism. Once recruited, TOPBP1 uses its ATR Activation Domain (AAD) to physically interact with ATR, and its binding partner ATRIP, thereby activating ATR kinase (Kumagai et al., 2006). Another physical interaction that occurs at sites of damage is between TOPBP1 and the RAD9 component of the trimeric DNA clamp protein 911 (RAD9, RAD1, and HUS1). TOPBP1 uses its BRCT1&2 domains to bind RAD9 (Delacroix et al., 2007, Lee et al., 2007, Rappas et al., 2011), and while this interaction is dispensable for ATR activation per se, binding of TOPBP1 to RAD9 is required for activated ATR to access some, but not all, of its substrates (Lupardus and Cimprich, 2006). Another factor that interacts with TOPBP1 at sites of damage is RHINO, which plays a stimulatory role in ATR signaling (Cotta-Ramusino et al., 2011, Lindsey-Boltz et al., 2015). Both RAD9 and RHINO bind the same site on TOPBP1 (BRCT1; Day et al., 2018), and thus it is unclear how these factors may be present together, at the same time, during ATR signaling.

One emerging idea is that TOPBP1’s role in ATR signaling involves its ability to oligomerize. This was suggested by recent work showing that forced “dimerization” of murine TOPBP1, using the FKBP dimerization module, allows ATR activation in the absence of exogenously applied DNA damage (Zhou et al., 2013). This was an interesting finding as one of the great mysteries surrounding TOPBP1 is why it is only competent to activate ATR when the two are together, on DNA, at sites of damage. Early studies showed that a recombinant protein comprised of GST fused to the TOPBP1 AAD could potently activate ATR when added to Xenopus egg extracts lacking DNA, showing that soluble ATR is competent for activation (Kumagai et al., 2006). Therefore, it is soluble TOPBP1 that is incompetent for activation, and thus TOPBP1 must change in some manner when on DNA to allow activation. One possibility is that this change involves oligomerization.

In this study, we examine TOPBP1 oligomerization with an eye towards understanding how oligomerization impacts TOPBP1’s role in ATR signaling. Results show that TOPBP1 oligomerizes via homotypic interaction between the BRCT 1&2 domains and heterotypic interaction between BRCT1&2 and BRCT4&5. We also address how TOPBP1 oligomers behave when challenged with a heterologous binding partner, and whether TOPBP1 oligomers can simultaneously interact with more than one BRCT1&2 binding factor at the same time. Lastly, we provide evidence that the TOPBP1 oligomeric state important for ATR activation is that of a tetramer.

2. Material and Methods

2.1. Materials

2.1.1. Plasmids

The expression vector to produce GST-RAD9 Tail has previously been described (Duursma et al., 2013). All other plasmids used in this study are described below, in table format, and all are Xenopus proteins. Specific details about plasmid construction are available upon request.

Plasmid Name Vector AA coordinates
MBP-TOPBP1 AK66 pUC57-MBP 1–1513
6Xmyc-TOPBP1 Cut5 pCS2+MT 1–1513
6Xmyc-NT TOPBP1 pMM2 pCS2+MT 1–758
6Xmyc-CT TOPBP1 pHG131 pCS2+MT 760–1513
6Xmyc-RAD9 pAK45 pCS2+MT 1–377
6xmyc-RAD9 Tail pKM29 pCS2+MT 270–377
6Xmyc-RHINO pKM13 pCS2+MT 1–252
MBP-BRCT1&2 E.coli expression vector pAK20 pMal-c5x 91–275
T7-BRCT1&2 E. coli expression vector pKM68 pET28A 91–275
GST-BRCT4&5 E.coli expression vector pHG49 pGEX-4T3 480–758
MBP-BRCT4&5 E. coli expression vector pAK21 pMal-c5x 480–758
MBP-RAD9 E.coli expression vector pAK46 pMal-c5x 1–377
MBP-RHINO E.coli expression vector pKM27 pMal-c5x 1–252
GST-AAD E.coli expression vector pHG117 pGEX-4T3 972–1279
MBP-AAD E.coli expression vector pHS40 pMal-c5x 972–1279
MBP-FKBP-AAD E.coli expression vector pHS47 2XFKBP-pMal-c5x 972–1279
6Xmyc-BRCT0 pHG126 pCS2+MT 1–99
6Xmyc-BRCT1 pHG129 pCS2+MT 100–191
6Xmyc-BRCT2 pJA23 pCS2+MT 191–333
6Xmyc-BRCT3 pJA18 pCS2+MT 333–480
6Xmyc-BRCT4 pJA24 pCS2+MT 527–622
6Xmyc-BRCT5 pJA25 pCS2+MT 627–800
6Xmyc-BRCT6 pAK15 pCS2+MT 894–970
6Xmyc-BRCT7 pAK17 pCS2+MT 1235–1350
6Xmyc-BRCT8 pAK18 pCS2+MT 1345–1470
6Xmyc-BRCT0–2 pHG128 pCS2+MT 1–333
6Xmyc-BRCT4&5 pJA19 pCS2+MT 480–758
6Xmyc-TOPBP1 W171R pAK22 pCS2+MT 1–1513
6Xmyc-TOPBP1 W265R pAK27 pCS2+MT 1–1513
6Xmyc-TOPBP1 W426R pHS2 pCS2+MT 1–1513
6Xmyc-TOPBP1 W603R pHG26 pCS2+MT 1–1513
6Xmyc-TOPBP1 W708R pHG27 pCS2+MT 1–1513
6Xmyc-TOPBP1 W966R pHS17 pCS2+MT 1–1513
6Xmyc-TOPBP1 Y1326R pHG55 pCS2+MT 1–1513
6Xmyc-TOPBP1 W1462R pHG51 pCS2+MT 1–1513
6Xmyc-TOPBP1 K155E pAK85 pCS2+MT 1–1513
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2.1.2. Recombinant proteins

The recombinant proteins used in this study were MBP-TOPBP1 BRCT1&2, T7-TOPBP1 BRCT1–2, GST-TOPBP1 BRCT4&5, MBP-TOPBP1 BRCT 4–5, MBP-TOPBP1 AAD, GST-TOPBP1 AAD, MBP-FKBP-TOPBP1 AAD, MBP-RAD9, GST-RAD9 Tail, and MBP-RHINO. All proteins were expressed in E. coli BL21(DE3) cells, at 37°C for 4 hours, and all proteins were purified from the soluble fraction according to standard procedures (detailed below).

2.1.3. Antibodies

We used the following commercially sourced antibodies in this work: Myc (Millipore Sigma #M4439), MBP (New England Biolabs #E8032S), GST (Millipore Sigma #05–782), T7 (Millipore Sigma #69522), CHK1 (Santa Cruz Biotechnology #sc-8408), P-CHK1 (Cell Signaling Technology #2341S), thyroglobulin (Santa Cruz Biotechnology #sc-53543), and aldolase (GeneTex #GTX101408). We also used our own antibody against Xenopus TOPBP1, HU142, which has been described (Van Hatten et al., 2002).

2.2. Methods

2.2.1. Xenopus egg extracts

Extracts were prepared and immunodepletion of TOPBP1 was performed as previously described (Van Hatten et al., 2002, Yan et al., 2006, Gillespie et al., 2012).

2.2.2. Sucrose density gradient centrifugation

Sucrose gradients (1.4 ml) were formed by layering 200 μl each of 10%, 15%, 20%, 25%, 30%, 35%, and 40% sucrose in egg lysis buffer salts (2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES-KOH pH 7.7) and incubating for 2 hr at room temperature and 1 hr at 4 °C. Samples were overlayed onto the gradients and centrifuged at 30,000 rpm for 16 hr at 4 °C in a TLS55 rotor in a Beckman TL100 ultracentrifuge. Fractions were collected via bottom puncture of the tubes with a 21-guage needle. Molecular size standards were human thyroglobulin (GeneTex #GTX14718) and rabbit aldolase (Millipore Sigma A2714–500U).

2.2.3. IVTT production of proteins and determination of protein concentrations

IVTT reactions were performed using the SP6 TnT® Quick Coupled Transcription/Translation System (Promega #L2080) according to the manufacturer’s instructions. To determine the concentration of myc-TOPBP1 and MBP-TOPBP1 produced in a typical IVTT reaction the samples were probed with antibody HU142, which recognizes Xenopus TOPBP1 (Van Hatten et al., 2002), and the signal intensity was compared to a dilution series of Xenopus egg extract. The concentration of TOPBP1 in Xenopus egg extract is known (it is 37.4 nM, see Wühr et \al., 2014) and thus by matching Western blot signal intensities between egg extract and the IVTT reaction we could obtain a reliable estimation of the concentrations of IVTT-produced TOPBP1 derivatives. To estimate the concentration of myc-RAD9 we probed IVTT reactions with anti-myc antibody and compared the signal intensity to those obtained for myc-TOPBP1, whose concentration was known.

2.2.4. Recombinant Protein Expression and Purification

Plasmids were transformed in BL21 (DE3) Competent E. Coli cells and grown in LB media (GST fusion proteins) or LB media with 0.2% glucose (MBP fusion proteins) with ampicillin. At an OD600 of 0.6–0.8, cells were induced with 0.3 mM IPTG for 4 hours at 37 °C. Cells were then harvested by centrifugation (4000 rpm for 20 min) and washed with phosphate-buffered saline (PBS). Cells were lysed by sonication in GST column buffer (40% PBS, 60% TEN buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0, 100 mM NaCl) supplemented with cOmplete protease inhibitor cocktail tablets (Roche #04693159001) and 10 mg lysozyme for GST fusion proteins or in maltose column buffer (20 mM Tris-HCl, pH 7.4, 0.3 M NaCl, and 1 mM EDTA) with 1mM DTT and cOmplete protease inhibitor cocktail tablets for MBP fusion proteins. Lysates were cleared by centrifugation (15,000 rcf for 20 min) and incubated with glutathione sepharose (BioVision #6566) for GST fusion proteins or amylose resin (New England Biolabs #E8021S) for MBP fusion proteins for 1 hour at 4 °C. Beads were poured over a column and washed with column buffer. GST fusion proteins were eluted with 20 mM glutathione in 75 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.1% Triton-X. MBP fusion proteins were eluted with 10 mM maltose in maltose column buffer. Proteins were dialyzed overnight into PBS.

2.2.5. Protein binding assays

Three kinds of protein binding assays were utilized. The first involved incubation of IVTT proteins with a large excess of recombinant GST or MBP fusion proteins purified from E. coli, in what we refer to as a “pull-down” assay. For these assays 10–20 ul of IVTT proteins were mixed with 5–10 ug of GST or MBP fusion protein and incubated for 60 minutes at 30°C. The mixtures were diluted into 400 ul of Binding Buffer (PBS + 0.1% NP-40) and then incubated with the appropriate resin (amylose for MBP, New England Biolabs # E8021S; glutathione sepharose for GST, BioVision #6566) for one hour at 4°C. The beads were washed four times with 500 ul of Binding Buffer and then eluted with 2X Sample Buffer (Millipore Sigma #S3401–10VL). The input and bound fractions were then analyzed via Western blotting using standard conditions.

The second type of binding assay, co-precipitation, involved co-incubation of IVTT produced proteins followed by capture on amylose resin. For these assays MBP-tagged TOPBP1 IVTT protein was mixed with target myc-tagged IVTT protein at a 1:1 ratio and incubated at 30°C for 60 minutes. Samples were then diluted into 400 ul of Binding Buffer and mixed with amylose resin for one hour at 4°C. The samples were then processed as described above for the pull-down assay.

The third type of binding assay (done once, in Figure 3E) was a variant of the pull-down where two recombinant proteins purified from E. coli were mixed, at 10 ug each, in PBS and then treated as described above for the pull-down assays using IVTT proteins.

Figure 3. TOPBP1’s BRCT1&2 binds itself and BRCT4&5.

Figure 3.

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A. Pull-down assay with MBP-BRCT1&2 and the indicated myc-tagged individual BRCT domain. Samples were processed as in Figure 1B. Inputs correspond to 3.4% of the starting material. Data shown are representative of two independent experimental replicates. B. Same as (A) except MBP-BRCT4&5 was used. C. Pull-down assay with MBP and MBP-BRCT1&2 and the indicated myc-tagged individual BRCT domain. Samples were processed as in Figure 1B. Inputs correspond to 3.4% of starting material. Data shown are representative of two independent experimental replicates. D-E. The indicated MBP fusion proteins were used in a pull-down assay with myc-tagged BRCT1&2 or BRCT4&5. Samples were processed as in Figure 1B. Inputs correspond to 3.4% of the starting material. Data shown are representative of two independent experimental replicates. F. Pull-down assay with the indicated recombinant proteins, all of which were expressed and purified from E. coli. Inputs correspond to 2.5% of the starting material. Data shown are representative of two independent experimental replicates. G. Western blot of recombinant T7-tagged BRCT1&2 that had been optionally treated with the EDC/NHS crosslinking reagent, as detailed in the Methods. Data shown are representative of two independent experimental replicates. H. Same as (G) except purified GST was used, at the indicated concentration. I. Pull-down assay with recombinant, purified MBP-BRCT4&5, myc-tagged BRCT0–2, and the indicated molar excess (over myc-BRCT0–2) of GST or GST-RAD9 Tail. Data shown are representative of two independent experimental replicates.

2.2.6. Protein crosslinking

Protein, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, and N-hydroxysuccinimide were combined at a 1:10:25 molar ratio in PBS at pH5. The solution was then incubated at room temperature for 4 hours with gentle agitation every hour. After 4 hours, a portion of the sample was diluted 1:10 in 2xSB and analyzed by Western blotting.

2.2.7. Gel filtration chromatography

Samples were fractionated on a Superdex 200 16/60 column at 0.9 ml/minute flow rate in PBS + 0.1% Triton buffer using Bio-Rad’s Biologic Duo Flow instrument. Fractions were collected and analyzed by SDS-PAGE followed by Coomassie blue staining.

3. Results

3.1. TOPBP1 oligomerizes in a manner that allows binding of heterologous partners

Previous work has shown that human TOPBP1 can form oligomers, as demonstrated by co-immunoprecipitation of differentially tagged forms of the protein (Liu et al., 2014). To examine this property for Xenopus TOPBP1 we chose to perform binding assays using the maltose binding protein (MBP) system. For this, MBP fusion proteins are used as prey and protein complexes that form on MBP fusion proteins are purified using amylose beads, which bind to MBP. We first wanted to be sure that neither TOPBP1 nor RAD9, which we use as a positive control in TOPBP1 binding assays, would efficiently bind to MBP. We therefore did binding assays with recombinant MBP, or an MBP-RAD9 fusion, and tested for interaction with myc-tagged TOPBP1 or myc-tagged RAD9 (Figure 1A). The myc-tagged proteins were produced by in vitro transcription and translation (IVTT) in rabbit reticulocyte lysates. After IVTT, recombinant MBP proteins were added to the lysates and incubation was continued for 60 minutes. The samples were then diluted in binding buffer and the mixtures were incubated with amylose beads, the beads were washed, and bound proteins eluted and analyzed by Western blotting. As shown in Figure 1B, both myc-TOPBP1 and myc-RAD9 could bind to MBP-RAD9, as expected (RAD9 self interacts, see Takeshi et al., 2015), but neither protein could bind to MBP alone. We conclude that neither TOPBP1 nor RAD9, nor the myc tags themselves, can efficiently bind to either MBP or the amylose beads.

Figure 1. Biochemical analysis of TOPBP1 oligomers.

Figure 1.

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A. Schematic representation of the various tagged forms of TOPBP1 and RAD9 that were analyzed. B. A pull-down assay where recombinant MBP and MBP-tagged RAD9 were purified from E. coli and added to IVTT-produced myc-tagged full length TopBP1 and RAD9. After the binding reaction, samples were diluted and incubated with amylose beads (which bind MBP). Samples were washed, eluted, and analyzed by Western blotting. The blots labeled “bound” represent material eluted off the amylose beads. The blot labeled “input” represents 3.4% of the myc-tagged proteins in the binding reaction. Data shown are representative of two independent experimental procedures. C. Co-precipitation assay where IVTT-produced MBP-TOPBP1 (full-length, FL) was mixed with the indicated IVTT-produced myc-tagged proteins. After incubation, the samples were precipitated using amylose beads and co-precipitating proteins were analyzed via Western blotting. The blots labeled “bound” represent material eluted off the amylose beads. The blot labeled “input” represents 3.4% of the input into the binding reactions. Data shown are representative of two independent experimental replicates. D. A pull-down assay where recombinant GST-RAD9 Tail (GST-RAD-T) was purified from E. coli and used together with IVTT-produced and myc-tagged TOPBP1 BRCT0–2. CK2i was optionally added prior to addition of GST-RAD9 Tail to the binding reaction. Samples were processed as in B except samples were incubated with glutathione beads (which bind GST). The blot labeled “input” represents 3.4% of the input into the binding reactions. Data shown are representative of two independent experimental replicates. E. A pull down assay where GST and GST-Rad9 Tail are used with myc-tagged and IVTT-produced full-length TopBP1 and TopBP1 BRCT 0–2. Samples were processed as in D. The blot labeled “input” represent 3.4% of the input into the binding reactions. F-G. Co-precipitation assays were performed with MBP-TOPBP1 and myc-tagged RAD9 (F) or myc-tagged TOPBP1 (G) in the presence of the indicated molar excess of GST or GST-Rad9 Tail (GST-R9T). The samples were processed as in Figure 1C. Data shown are representative of two independent experimental replicates. H. TOPBP1 was produced via IVTT in rabbit reticulocyte lysates and then centrifuged through a sucrose density gradient as described in the Methods.

We next used the MBP binding assay to examine the self-interaction capacity of TOPBP1. For this we produced MBP-TOPBP1 together with myc-tagged TOPBP1 (~180 kDa), two myc-tagged fragments of TOPBP1 (each ~90 kDa), and the positive control, myc-tagged RAD9 (Figure 1A). All of the proteins were produced by IVTT, and after IVTT the lysates were mixed together and incubated for an hour at 30°C. To assess binding the samples were then diluted and bound to amylose beads. The beads were then washed and the samples eluted prior to Western blotting. As shown in Figure 1C, myc-TOPBP1 was co-precipitated with MBP-TOPBP1, showing that the two proteins interact and thus that TOPBP1 oligomerizes. One may wonder how the MBP- and myc-tagged forms of TOPBP1 can interact if they themselves are oligomers, and this is easily explained by the ability of TOPBP1 protomers to exchange with one another during the binding reaction. We next qualitatively compared the interaction of TOPBP1 with itself to how it interacts with RAD9, by performing a similar binding assay with MBP-TOPBP1 and myc-RAD9. As shown in Figure 1C, MBP-TOPBP1 interacts with myc-RAD9 with a similar efficiency as myc-TOPBP1, as assessed by the signal intensities in the bound fractions on the blot (note that input levels of myc-TOPBP1 and myc-RAD9 are similar). Lastly, we divided TOPBP1 into N- and C-terminal fragments and subjected these to binding assays with MBP-TOPBP1. The N-terminal fragment could interact well with MBP-TOPBP1, and we also detected binding with the C-terminal fragment. We conclude that Xenopus TOPBP1 can self-associate and, given that the non-overlapping N- and C-terminal fragments each bound to MBP-TOPBP1, that there are multiple determinant(s) for this self-association.

Having established a simple and robust assay for TOPBP1 oligomerization we went on to use this assay to delineate some basic properties of the TOPBP1 oligomer. The first question we asked was will the oligomer bind heterologous binding partners, or does TOPBP1 transition between the oligomer and a monomeric form that uniquely interacts with heterologous partners? As a model heterologous binding partner we chose a GST protein fused to the “tail” domain of RAD9 (GST-RAD9 Tail, see Duursma et al., 2013). The RAD9 tail is a region of the protein that is constitutively phosphorylated by casein kinase 2 (CK2) in a manner that allows it to bind TOPBP1’s BRCT1&2 domains (Takeishi et al., 2010). Fortunately, rabbit reticulocyte lysates contain active CK2 (Hathaway and Traugh, 1979), but we wanted to be sure that the interaction between TOPBP1’s BRCT1&2 domains and GST-RAD9 Tail is CK2 dependent, and thus physiologically relevant. For this, we set up binding assays and optionally included a small molecule inhibitor of CK2 (CX-4945, referred to as CK2i, see Fergusuon et al., 2011). The binding partners for this experiment were GST-RAD9T and a myc-tagged TOPBP1 fragment corresponding to BRCT domains 0–2 (myc-BRCT0–2). After IVTT of myc-BRCT0–2 the lysates were optionally treated with CK2i followed by addition of GST-RAD9 Tail. The samples were incubated for 60 minutes and then diluted and incubated with glutathione sepharose beads, to capture GST-RAD9 Tail containing complexes. After washing, bound proteins were eluted and analyzed via Western blotting. As shown in Figure 1D, addition of CK2i blocked the interaction between GST-RAD9 Tail and myc-BRCT0–2, showing that binding is dependent on CK2. We also performed a control experiment where binding of myc-TOPBP1 or myc-BRCT0–2 was assessed for GST alone, relative to GST-RAD9 Tail. As expected, there was no detectable interaction between GST alone and either of the myc-tagged proteins (Figure 1E). We conclude that our binding assays with GST-RAD9 Tail faithfully recapitulate the interaction between the RAD9 Tail domain and TOPBP1’s N-terminus.

To pursue these observations we next asked if addition of an excess amount of GST-RAD9 Tail would disrupt TOPBP1 oligomerization, as would be the case if only monomers can bind heterologous partners like RAD9. We first determined the concentration of IVTT-produced proteins such as myc-RAD9, myc-TOPBP1, and MBP-TOPBP1, and found that they are present at ~90 nM in our IVTT system (see Materials and Methods). We next asked if addition of 22- or 100-fold excess of GST-RAD9 Tail to a co-precipitation assay with myc-RAD9 and MBP-TOPBP1 would inhibit binding of RAD9 to TOPBP1. We found that it did, quite effectively, whereas 100X excess of GST alone had no effect (Figure 1E). This shows that our approach is valid. We next asked if 22X or 100X GST-RAD9 Tail would inhibit TOPBP1 oligomerization and found that it did not, as myc-TOPBP1 and MBP-TOPBP1 could still interact with one another despite the large excess of GST-RAD9 Tail present in the assay (Figure 1F). Thus TOPBP1 can still form oligomers in the presence of a high concentration of heterologous binding partner, and based on this we conclude that binding of heterologous partners to TOPBP1 does not come at the expense of oligomerization.

The binding assay in Figure 1C shows that TOPBP1 oligomers are not rigidly held together; rather, it appears that TOPBP1 transitions between monomer and oligomer readily, as otherwise binding of MBP-TOPBP1 to myc-TOPBP1 would be difficult to observe. It was thus of interest to determine the steady-state distribution of TOPBP1 monomer/oligomer in our system. For this we performed an IVTT reaction with myc-TOPBP1 and analyzed its molecular mass via sucrose density centrifugation, in conjunction with two size standards, thyroglobulin (660 kDa), and aldolase (144 kDa). Monomers of the ~180 kDa TOPBP1 will sediment near aldolase in the gradient, with oligomers running below aldolase and towards the thyroglobulin marker. As shown in Figure 1G, while some monomeric TOPBP1 is clearly present, the majority of the protein sediments in a manner consistent with oligomers. It is possible that the TOPBP1 in the lower fractions is brought there via binding to heterologous partners, however this is unlikely as the lysates used for IVTT are made from reticulocytes, cells that do not have nuclei. TOPBP1 is exclusively found in the nucleus (Yamane et al., 2002), and thus few, if any, of its natural binding partners would be present in the IVTT reactions. We conclude that TOPBP1 can exist, at steady state, in a monomeric form and also as an oligomer. The nature of these oligomers is not yet known, however the data in Figure 1F suggest that dimers, trimers, and tetramers are all possible.

3.2. BRCT2 is required for TOPBP1 oligomerization

Previous work has shown that BRCT-containing proteins can oligomerize via direct interaction between their BRCT domains. Three well-studied examples of BRCT-BRCT interactions include the XRCC1-DNA ligase III DNA repair complex, the tumor suppressor BRCA1 protein, and the yeast checkpoint proteins Rad9/Crb2. Both XRCC1 and DNA ligase III (Lig3) homodimerize via the self-association of their BRCT domains, and, furthermore, a heterotetramer of XRCC1 and Lig3 dimers will form via interaction of XRCC1 BRCT2 and the sole BRCT domain of Lig3 (Cuneo et al., 2011). For BRCA1, recent work has shown that interaction of the BRCA1 BRCT domains with phosphorylated Abraxas triggers BRCT-BRCT dimer formation in a manner that involves extensive contacts between the BRCT domains (Wu et al., 2016). Finally, Rad9 (budding yeast) and Crb2 (fission yeast) are checkpoint adaptor proteins that dimerize via their BRCT domains (Du et al., 2004, Granata et al., 2010, Soulier and Lowndes, 1999). We therefore predicted that TOPBP1 oligomerization would involve BRCT-BRCT contacts, and to test this we made a library of point mutants where individual BRCT domains were inactivated via mutation of a highly conserved hydrophobic residue within α-helix 3 (Figure 2A). This residue, tryptophan in BRCTs 1–6 and tyrosine in BRCTs 7&8 (BRCT 0 lacks such a residue), is buried deep within the hydrophobic core of the globular BRCT domain and substitution to a charged residue such as arginine is likely to have a major impact on folding of the domain. Indeed, mutation of this conserved tryptophan within the XRCC1 BRCT2 domain prevents its interaction with the BRCT domain of Lig3 (Dulic et al., 2001). We tested the ability of each W/Y->R mutant to bind MBP-TOPBP1 (Figures 2B-E) and found that W265R, located in BRCT2, showed a near-complete reduction in binding, and that W171R (BRCT1) showed a reduction in binding (Figure 2B). This shows that BRCT1&2 is important for TOPBP1 oligomerization. This result also shows that the interaction between MBP-TOPBP1 and wild type myc-TOPBP1 that we have observed is specific, and thus is not due to non-specific protein aggregation. Having observed that BRCT2 is necessary for TOPBP1 oligomerization we next asked if the BRCT1&2 region is sufficient for binding to TOPBP1. For this we made a recombinant protein consisting of MBP fused to TOPBP1’s BRCT 1&2 domains and asked if it could bind full-length myc-TOPBP1. As shown in Figure 2F, MBP-BRCT 1&2 could bind myc-TOPBP1. These results highlight the importance of the BRCT1&2 region in promoting TOPBP1 oligomerization.

Figure 2. Mutational analysis of BRCT-domain requirements for TOPBP1 oligomerization.

Figure 2.

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A. Schematic representation of the positions of the highly conserved aromatic residues within TOPBP1 BRCT domains 1–8. B-E. Co-precipitation assays with IVTT-produced MBP-TOPBP1 binding to the indicated IVTT-produced and myc-tagged TOPBP1 point mutants. Samples were processed as in Figure 1C. Inputs correspond to 3.4% of the starting material. Data shown are representative of two independent experimental replicates. F. Pull-down assays with MBP or MBP-BRCT1&2 and myc-tagged TOPBP1. Samples were processed as in Figure 1B. Inputs correspond to 3.4% of the starting material. Data shown are representative of two independent experimental replicates.

3.3. BRCT 1&2 binds directly to itself and to the BRCT 4&5 domains

We next sought to determine which, if any, of TOPBP1’s BRCT domains could bind to BRCT 1&2. Individual domains were myc tagged and then expressed by IVTT prior to binding assays with recombinant MBP-BRCT 1&2. As shown in Figure 3A, BRCT domains 1,2, and 5 could bind well to MBP-BRCT 1&2. Given that BRCT5 scored positive for binding, we next asked which of TOPBP1’s BRCT domains could bind to an MBP-BRCT 4&5 fusion protein and found that only BRCTs 1 and 2 bound well (Figure 3B). These results suggest two interactions for BRCT 1&2, one homotypic and the other heterotypic with BRCT 4&5. It was formally possible that BRCT domains 1, 2, and 5 bind to MBP and not the BRCT portion of the fusion protein, however a direct test ruled this possibility out (Figure 3C). To pursue these observations we next examined binding of these domains in the context of BRCT pairs, and found that MBP-BRCT1&2 could bind myc-BRCT1&2, and also myc-BRCT4&5, whereas MBP-BRCT4&5 could bind myc-BRCT1&2 but not myc-BRCT4&5. MBP alone did not bind any of the myc-tagged proteins (Figures 3D&E).

Our data identify two new interactions for TOPBP1, BRCT1&2::BRCT1&2 and BRCT1&2::BRCT4&5, and we next wanted to determine if these are direct interactions, or if they are bridged by an unknown factor present in the IVTT reactions. For the BRCT1&2::BRCT4&5 interaction, we used two recombinant proteins that were expressed and purified from E. coli, MBP-BRCT1&2 and GSTBRCT4&5. As shown in Figure 3F, GST-BRCT4&5, but not GST alone, could bind MBP-BRCT1&2. This results shows that the BRCT1&2::BRCT4&5 interaction is direct and does not require any post-translational modification (PTMs, which E. coli cannot do). We next examined the BRCT1&2 self-association, and for this we performed crosslinking experiments using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS; Grabarek and Gergely, 1990) to crosslink a 6His- and T7-tagged BRCT1&2 recombinant protein purified from E. coli. When T7-BRCT1&2 was analyzed we saw that treatment with EDC/NHS produced a unique band that migrated at ~120 kDa after SDS-PAGE (Figure 3G). Given that the T7-BRCT1&2 monomer is ~30 kDa it appears that BRCT1&2 can self-associate to form a tetramer. We note that the crosslinking efficiency was low, as inferred by the ratio of tetramer to monomer in the sample, and we suspected that this is because the input concentration of T7-BRCT1&2 was low (5 uM). The T7-BRCT1&2 protein precipitates at concentrations higher than 5 uM, thus limiting the amount of material we could use. To confirm this suspicion we examined crosslinking efficiency for a natural dimer, GST (McTigue et al., 1995), and we found that crosslinking efficiency was indeed poor at the 5 uM concentration, relative to higher concentrations (Figure 3H). Based on these data, we conclude that both the BRCT1&2::BRCT1&2 and BRCT1&2::BRCT4&5 interactions are direct and occur independently of PTMs.

It is noteworthy that the BRCT 1&2 region of TOPBP1 is involved in oligomerization, given its dominant role in mediating interaction with heterologous binding partners. It was therefore of use to ask if the BRCT::BRCT interactions we have defined in this work can be disrupted by binding of heterologous binding partners. For this we again performed a competition experiment with excess GST-RAD9 Tail and asked if binding of BRCT0–2 to BRCT4&5 would be sensitive to the presence of excess GST-RAD9 Tail. As shown in Figure 3I, this was not the case. Thus, like oligomerization of the full-length proteins (Figure 1E), oligomers composed of BRCT0–2 and BRCT4–5 readily form in the presence of heterologous BRCT1&2 binding partners.

3.4. Different BRCT1&2 binding partners can simultaneously interact with a TOPBP1 oligomer

We continued our study of TOPBP1 oligomerization by asking if a TOPBP1 oligomer could simultaneously bind two different heterologous partners through its BRCT1&2 region. This was of interest as, if simultaneous binding is allowed, then the capacity for TOPBP1 to serve as a scaffold protein is greatly enhanced. For this we used GST-Rad9 Tail and another TOPBP1 BRCT1&2 binding partner, RHINO (Figure 4A). Recent work has shown that the RAD9 Tail and RHINO both bind to TOPBP1 BRCT1 in a lysine 155 (K155) dependent manner (Day et al., 2018). K155 is part of the phosphate-binding pocket of BRCT1 and mutation of K155 to glutamic acid blocks binding of phosphorylated substrates to the pocket (Day et al., 2018, Rappas et al., 2011). Indeed, using pull-down assays, we observed that wild type TOPBP1, but not the K155E mutant, could bind RAD9 (Figure 4B) and the same was true for RHINO (Figure 4C). These data show that both RAD9 and RHINO have single binding sites within TOPBP1, and it is the same site, BRCT1. If a TOPBP1 oligomer can bind both RAD9 and RHINO at the same time then MBP-tagged RHINO should be able to co-precipitate myc-tagged RAD9 Tail, in a TOPBP1-dependent manner (Figure 4A). Importantly, previous work has shown that while RAD9 and RHINO interact (Cotta-Ramusino et al., 2011, Lindsey-Boltz et al., 2015), RHINO binds to the core domain of RAD9 and not the tail (Lindsey-Boltz et al., 2015). Therefore, any co-precipitation of the RAD9 Tail by MBP-RHINO would be through a bridging effect of the TOPBP1 oligomer. As shown in Figure 4D, this is what we observed, as inclusion of TOPBP1 allowed RHINO to form a complex with the RAD9 Tail. We also performed the reciprocal experiment, where we asked if GST-tagged RAD9 Tail could co-precipitate myc-tagged RHINO in a TOPBP1-dependent manner and found that it could (Figure 4E). We note that while some myc-RHINO is seen in the sample lacking TOPBP1, possibly due to low-level binding of myc-RHINO to GST, the signal was clearly stronger in the sample containing TOPBP1. Taken together, these data show that the TOPBP1 oligomer can simultaneously accommodate multiple BRCT1&2 binding partners.

Figure 4. TOPBP1 oligomers can simultaneously interact with two distinct BRCT 1 binding partners.

Figure 4.

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A. Schematic depicting the experimental question - when mixed together can TOPBP1, RHINO, and the RAD9 Tail (RAD9-T in the drawing) form a complex where RHINO and the RAD9 Tail are bridged by oligomeric TOPBP1? Note that TOPBP1 is depicted as a dimer for reasons of simplicity only. B-C. Pull-down assays with the indicated recombinant, purified MBP fusion protein and IVTT-produced and myc-tagged wild type (WT) or mutant (K155E) TOPBP1. Samples were processed as in Figure 3D-E. Input refers to 3.4% of the starting material. Data shown are representative of two independent experimental replicates. D. Pull-down assay with purified recombinant MBP-RHINO and IVTT-produced myc-RAD9 Tail and IVTT-produced and untagged TOPBP1. TOPBP1 was detected with antibody HU142. Samples were processed as in Figure 1B. Input refers to 3.4% of the starting material. Data shown are representative of two independent experimental replicates. E. Same as (D) except purified recombinant GST-RAD9 Tail and myc-tagged and IVTT-produced RHINO were used in place of MBP-RHINO and myc-RAD9 Tail, respectively.

3.5. The TOPBP1 AAD is active as a tetramer

In a final set of experiments we sought to address the functional consequences for TOPBP1 oligomerization, and we focused on the oligomeric state required to activate the ATR kinase. Previous work has shown that forced “dimerization” of TOPBP1 triggered ATR activation without application of exogenous DNA damage (Zhou et al., 2013). In these experiments, TOPBP1 was fused to the FKBP dimerization module (Clackson et al., 1998). FKBP modules will dimerize in the presence of the synthetic ligand AP20187, and this previous work showed that FKBP-TOPBP1 fusion proteins would activate ATR, in a ligand-dependent manner, in undamaged cells. A simple interpretation of these data is that monomeric TOPBP1 cannot activate ATR but dimeric TOPBP1 can. Importantly, however, the true oligomeric state of TOPBP1 was not addressed in these experiments, and thus it was unclear if the AP20187 ligand was inducing dimers, or more higher-order oligomers. Complicating things further is other work showing that the human TOPBP1 AAD, when isolated from the rest of the protein, has the ability to form dimers (Thada and Cortez, 2019). To examine how multimerization of the TOPBP1 AAD might impact ATR activation we produced two recombinant fusion proteins, GST-TOPBP1 AAD and MBP-TOPBP1 AAD. When added to Xenopus egg extracts, the GST-AAD protein could readily promote ATR activation, as assessed by the appearance of phosphorylated CHK1 (P-CHK1), a key ATR substrate (Figure 5A, see Guo et al., 2000). This is consistent with previous results (Kumagai et al., 2006). By contrast, the MBP-AAD protein was not nearly as active (Figure 5A). One difference between GST and MBP is that GST is a dimer whereas MBP is a stable monomer (McTigue et al., 1995, Reuten et al., 2016). We therefore analyzed both recombinant proteins by gel filtration chromatography, to gain insight into their oligomerization status. The GST-AAD monomer is ~72 kDa, while MBP-AAD is larger, ~92 kDa (Figure 5B). Gel filtration chromatography revealed that both proteins oligomerize, with MBP-AAD existing mostly as a dimer, based on its elution profile relative to molecular weight standards (Figure 5C). By contrast, GST-AAD displayed a broader elution profile, with some of the sample clearly eluting as complexes larger than a dimer (Figure 5C). One interpretation of these results, that is consistent with the known properties of GST, MBP, and AAD, is that MBP-AAD dimerizes via sequences within the AAD and that GST-AAD forms tetramers via the combined effects of the GST and AAD dimerization potential (Figure 5D). If so, then one explanation for the difference in ability of GST- and MBP-AAD to activate ATR is that the AAD is active as a tetramer but not as a dimer.

Figure 5. The TOPBP1 AAD is active as a tetramer and inactive as a dimer.

Figure 5.

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A. The indicated recombinant AAD fusion proteins were incubated in Xenopus egg extract at the indicated concentrations for 60 minutes at room temperature. Samples were taken and assessed for ATR activation via Western blotting for P-CHK1. The total level of CHK1 was also probed, as a loading control. Data shown are representative of two independent experimental replicates. B. Coomassie-stained gel showing the size of the indicated recombinant AAD fusion protein. C. Coomassie-stained gel showing the elution profile of the indicated recombinant AAD fusion protein after gel filtration chromatography. The elution peaks for two molecular weight standards are indicated at the top. Data shown are representative of two independent experimental replicates. D. Cartoon describing a plausible model for why GST-AAD is more active than MBP-AAD and how the FKBP dimerization module could increase activity of MBP-AAD. E. The indicated recombinant AAD fusion proteins were incubated in egg extract at 1.6uM each and the dimerizer (dim.) compound, AP20187, was optionally added, at 10uM. After a one-hour incubation the samples were assessed for ATR activation as in part A. Data shown are representative of two independent experimental replicates. F. Co-precipitation assay with IVTT-produced MBP-TOPBP1 binding to the indicated IVTT-produced and myc-tagged wild type (WT) or point mutant W1138R. Samples were processed as in Figure 1C. Inputs correspond to 3.4% of the starting material. Data shown are representative of two independent experimental replicates.

To directly test this hypothesis we engineered an MBP-AAD derivative that contains the FKBP dimerization module (MBP-FKBP-AAD). In principle, ligand binding could bring together two MBP-FKBP-AAD dimers to make a tetramer, and this might allow more efficient ATR activation than is seen with MBP-AAD (Figure 5D). Equal amounts (1.6 uM) of GST-AAD, MBP-AAD, and MBP-FKBP-AAD were added to egg extract and ATR activation was assessed via probing for P-CHK1. As shown in Figure 5E, addition of ligand to the sample containing MBP-FKBP-AAD allowed the protein to activate ATR, and this was not observed in the sample containing MBP-AAD. We conclude that the AAD is most active when it is in a tetrameric form. This finding has implications for how full-length TOPBP1 activates ATR, as discussed below.

In a final experiment we tested the W1138R point mutant of TOPBP1 for its ability to bind MBP-TOPBP1. We did this because previous data has shown that this point mutant, located in the AAD, prevents TOPBP1 from activating ATR (Kumagai et al., 2006) and we and others have shown that the domain, at least when isolated away from the rest of the protein, can oligomerize (Figure 5C and Thada and Cortez, 2019 ). We found that W1138R did not impact TOPBP1 oligomerization, and thus the inability of this point mutant to activate ATR cannot be readily explained through oligomerization.

4. Discussion

The data presented here have revealed important biochemical properties of the critical ATR activator TOPBP1. Protein binding assays revealed that TOPBP1 self-associates, consistent with previous reports (Liu et al., 2014). Self-association is fluid, however, as when MBP-TOPBP1 and myc-TOPBP1 homo-oligomers are mixed together there is clearly exchange and MBP-TOPBP1/myc-TOPBP1 hetero-oligomers can readily form (Figure 1C). Consistent with this interpretation, we see by sucrose gradient sedimentation analysis that, at steady state, TOPBP1 exists as mixed bag of monomer and higher-order oligomers (Figure 1F). Thus it may be that there is no single oligomeric form that TOPBP1 assumes; rather, TOPBP1 may form a variety of oligomers, in a context-dependent manner. We discuss this possibility in more detail below.

TOPBP1 is presumed to function as a scaffold and, indeed, recent evidence suggests that TOPBP1 simultaneously interacts with RAD9 (via BRCT1) and the DDR factor 53BP1 (via BRCT2 and BRCT5, see Bigot et al., 2019). In the work presented here we asked two questions relevant to TOPBP1’s role as a scaffold. The first was does TOPBP1 interact with heterologous binding partners purely as a monomer, or can the oligomer bind heterologous partners? If it is only monomeric TOPBP1 that binds factors like RAD9 then we would expect that an excess of heterologous binding partner would disrupt TOPBP1 oligomers, however this was not the case (Figure 1E). TOPBP1 oligomers are thus capable of binding heterologous partners.

The second question was does the TOPBP1 oligomer simultaneously bind two distinct factors that occupy identical binding sites? This question is directly pertinent to how TOPBP1 interacts with RAD9 and RHINO during ATR signaling. Previous work has shown that RAD9 and RHINO both bind TOPBP1’s BRCT1 domain (Rappas et al., 2011; Day et al., 2018), and we have shown here, using the K155E mutant, that BRCT1 is the sole binding site for both proteins (Figures 4B&C). Furthermore, both RAD9 and RHINO participate in TOPBP1-mediated and ATR-directed phosphorylation of CHK1, with RAD9 being essential and RHINO playing a stimulatory role (Cotta-Ramusino et al., 2011; Lindsey-Boltz et al., 2015). Lastly, biochemical studies have shown that RHINO forms a stoichiometric complex with RAD9 (and RAD1 and HUS1 as well, see Lindsey-Boltz et al., 2015). These findings, taken together, presented a conundrum: we have two factors binding the same site on TOPBP1, and participating in the same signaling event, so how can this happen? We have resolved this conundrum with our finding that a TOPBP1 oligomer can simultaneously bind both RAD9 and RHINO (Figures 4D&E). This finding has implications for TOPBP1’s ability to organize different DDR factors during ATR signaling. Current models that envisage monomeric TOPBP1 suggest that TOPBP1’s multiple BRCT domains can bridge distinct factors via binding of these factors to distinct domains (Bigot e al., 2019). Indeed, recent work has shown that TOPBP1 can bridge RAD9 and 53BP1 via binding to BRCTs 1 (RAD9) and 2 and 5 (53BP1, see Bigot et al., 2019). Our finding that TOPBP1 can also bridge factors that occupy the same site on TOPBP1 thus significantly expands the capacity of the protein to serve as a scaffold.

Another important question concerning TOPBP1 oligomerization is how do the oligomers form? We report here that the BRCT 1&2 region plays a central role, as a point mutation in BRCT2 (W265R) prevents oligomerization (Figure 2B). Furthermore, we go on to show that BRCT1&2 can interact with itself and with BRCT4&5 (Figures 4A-C). Thus there are at least two points of self-contact for TOPBP1, and there are likely more, as we can detect binding of the C-terminal portion of TOPBP1, which lacks BRCTs 1&2 and 4&5, to full-length TOPBP1 (Figure 1C). The means by which TOPBP1 oligomerizes is likely complex, and distinct oligomers may form, in a context dependent manner. It is also possible that heterologous binding partners may shape TOPBP1 oligomers. For example, it is known that RHINO interacts strongly with RAD9 (Lindsey-Boltz et al., 2015), and thus binding of RHINO to RAD9 may strengthen the self-association of the BRCT 1&2 domains to create a more stable oligomer. In addition, PTMs may also help shape TOPBP1 oligomers. Previous work has shown that TOPBP1 can self-associate via the binding of its BRCT7&8 domains on one protomer to a phosphorylated residue (S1159 in the human protein) on another protomer (Liu et al., 2013). Lastly, we note that recent work has revealed a remarkable new function for TOPBP1 in holding broken chromosomes together during mitosis (Leimbacher et al., 2019). These studies suggest that TOPBP1 forms a filament that serves as a molecular glue to keep the chromosome together. Clearly we still have much to learn about the different structures and assembly mechanisms for TOPBP1 oligomers.

A final question concerning TOPBP1 oligomerization is what are the functional consequences? Previous work has shown that multimerization of TOPBP1 is likely to be important for how it activates ATR (Zhou et al., 2013). In this work we have analyzed the multimeric state of the AAD itself and we find that, indeed, the AAD must tetramerize in order to activate ATR in a soluble system (Figure 5). Thus one oligomeric state that is critical for TOPBP1 function is likely to be a tetramer. These data shed light on the long-standing mystery of why soluble TOPBP1 cannot efficiently activate ATR. One strong possibility is that the tetrameric form of TOPBP1 that is capable of activating ATR only forms at sites of damage. This could happen via the influence of heterologous binding partners, found only at sites of damage, that help to shape the tetramer. One prime candidate is RPA-ssDNA. Previous work from our laboratory has shown that TOPBP1 binds RPA-ssDNA in a manner that requires both RPA and ssDNA (Acevedo et al., 2016). Thus if RPA-ssDNA interactions help drive TOPBP1 assembly into tetramers then we have an explanation for why TOPBP1’s ability to activate ATR is restricted to sites of damage, as this is where RPA-ssDNA is found.

Highlights.

  • The ATR activator TOPBP1 oligomerizes via BRCT-BRCT domain interactions.

  • TOPBP1 oligomers can still bind heterologous binding partners.

  • The TOPBP1 AAD activates ATR as a tetramer.

Acknowledgments

This work was supported by NIH grant R01GM12287. We thank Karlene Cimprich for the gift of the GST-RAD9 Tail expression vector.

Funding Source

National Institutes of Health grant GM12287

Footnotes

Conflict of interest statement

The authors declare no conflict of interest.

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