Montales K et al. (2022), MRN-dependent and independent pathways for recr...

XB-ART-59242

PLoS One 2022 Aug 02;178:e0271905. doi: 10.1371/journal.pone.0271905.

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MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks.

Montales K , Ruis K , Lindsay H , Michael WM .

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Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing's tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.

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Species referenced: Xenopus laevis
Genes referenced: atm atr chek1 ctbp2 mdc1 mre11 nbn rad50 rbbp8 topbp1
GO keywords: DNA double-strand break processing [+]

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	Fig 1. Functional domains and binding partners of TOPBP1. Please see text for details. https://doi.org/10.1371/journal.pone.0271905.g001

	Fig 3. MRN is partially required for TOPBP1 recruitment to DSBs and ATR signaling. A. NBS1 antibodies were used to remove the MRN complex from XEE via immunodepletion. A mock-depleted sample was also prepared. The depleted extracts were then probed for the indicated proteins (panels “total XEE”). Note that NBS1 depletion results in the removal of all detectable NBS1, as well as MRE11, showing that the MRN complex has been removed. Note also that TOPBP1 is not co-depleted by the NBS1 antibody. The depleted extracts were then used for a DSB binding assay, and panels “DSB-bound” shows the occupancy of the indicated proteins on the DSB beads. The experiment shown is representative of two independently performed biological replicates. B. Same as (A) except CtIP occupancy was examined in this experiment. The experiment shown is representative of two independently performed biological replicates. C. NBS1 antibodies were used to remove the MRN complex from XEE via immunodepletion. A mock-depleted sample was also prepared. The depleted extracts were then used for a DMAX assay to asses ATR’s ability to phosphorylate its key substrate, CHK1. “λ DSBs”refers to phage lambda DNA that was digested with EcoRI and represents the source of DSBs for this experiment. DSBs were optionally added to the depleted extracts, and after incubation samples were pulled and probed for P-CHK1, to assess the ATR activity state, and unmodified CHK1, to ensure CHK1 was present in the extract, and NBS1, to assess the effectiveness of the immunodepletion. The experiment shown is representative of two independently performed biological replicates. The other replicate is shown in S2 Fig. Shown below the blots is quantification of P-CHK1. For both replicates, ImageJ software was used to quantify the signal intensity for the P-CHK1 samples. The values for the mock-depleted sample were set to 100, the values for the NBS1-depleted samples were adjusted accordingly, and the results are presented as a ratio of NBS1-depelted signal over mock-depleted signal. “Exp” stands for experiment. D. Same as (A) except CtIP was depleted in this experiment. The experiment shown is representative of two independently performed biological replicates. The CtIP antibody we used recognizes a background band in the total extract, that runs just below CtIP. The background band is denoted by two asterisks while the CtIP band is denoted by two dots. Note that the background band remains after CtIP depletion whereas all detectable CtIP is removed. https://doi.org/10.1371/journal.pone.0271905.g003
	Fig 4. Loss of ATM kinase activity promotes the accumulation of MRN and TOPBP1 on DSBs. A. A DSB binding assay was performed. One sample received magnetic streptavidin beads lacking DNA (empty beads) and the other two samples received DSB beads. Beads were incubated in XEEs treated with the indicated chemicals. ATMi is KU55933 at 100 uM. After incubation, samples were taken of the total extract and probed for the indicated proteins (panels “total XEE”). Please see the legend for Fig 3D for an explanation of the CtIP panel. Beads were isolated and washed and the probed for occupancy of the indicated proteins (panels “DSB-bound”). The experiment shown is representative of two independently performed biological replicates. B. A DSB-binding assay was performed in XEEs treated with the indicated compounds. Mirin was used at 100 uM. The experiment shown is representative of two independently performed biological replicates. https://doi.org/10.1371/journal.pone.0271905.g004
	Fig 5. ATM kinase activity is not essential for ATR signaling in the DMAX system. A. XEEs were treated with the indicated compounds and then λ DSBs were optionally added. After incubation samples were taken and probed for the indicated proteins. The P-CHK1 and P-ATM signals were then quantified, as in Fig 3C. ATRi was present at 100 uM, as was ATMi (KU55933). The experiment shown is representative of two independently performed biological replicates. B. Same as (A) except ATRi was omitted and two different ATMis were used, at the indicated concentrations. The experiment shown is representative of two independently performed biological replicates. https://doi.org/10.1371/journal.pone.0271905.g005
	Fig 6. The AT70 DNA structure requires MRN and ATM for ATR signaling. A. XEEs were incubated with either water, the A70 oligonucleotide, the T70 oligonucleotide, or the two oligonucleotides that had been annealed together (AT70). The samples were then probed for P-CHK1 and CHK1. The experiment shown is representative of two independently performed biological replicates. B. AT70 was optionally added to either mock- or NBS1-depleted extract. After incubation, samples were pulled and probed for the indicated proteins. The experiment shown is representative of two independently performed biological replicates. C. Extracts were treated with the indicated compounds and AT70 was optionally added. After incubation, samples were pulled and probed for the indicated proteins. ATMis were present at 100 uM. The experiment shown is representative of two independently performed biological replicates. D. The indicated DNAs at the indicated concentrations were added to XEEs and, after incubation, samples were pulled and probed for the indicated proteins. The experiment shown is representative of two independently performed biological replicates. https://doi.org/10.1371/journal.pone.0271905.g006
	Fig 7. Neither the 9-1-1 complex nor MDC1 are required for TOPBP1 recruitment to DSBs. A. A DSB binding assay was performed and the bound fraction was probed for the indicated proteins. The experiment shown is representative of two independently performed biological replicates. B. A DSB binding assay was performed and samples from total extract and the bound fraction was probed for the indicated proteins. For the total extract samples, 1 ul of XEE was loaded on the gel. Please see text for explanation of “matched exposures”. The experiment shown is representative of two independently performed biological replicates. C. A GST pull-down experiment was performed. The indicated GST fusion proteins were added to XEEs that had been treated with the indicated compounds and, after incubation, complexes were isolated back out of the extract via glutathione-sepharose beads and probed for the indicated proteins. Samples of the total extract were also probed for TOPBP1. The experiment shown is representative of two independently performed biological replicates. C. A DSB-binding assay was performed with XEEs treated with the indicated compounds at the indicated concentrations. Total extract and DSB-bound samples were then probed for the indicated proteins. The experiment shown is representative of two independently performed biological replicates. D. Same as (C) except a GST-MDC1 fusion protein was used in place of GST-RAD9 Tail. The experiment shown is representative of two independently performed biological replicates. https://doi.org/10.1371/journal.pone.0271905.g007
	Fig 8. Multiple pathways for TOPBP1 recruitment and ATR activation by DSBs in Xenopus. A. Please see text for details. B. Please see text for details. https://doi.org/10.1371/journal.pone.0271905.g008

References [+] :

Bakkenist, DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. 2003, Pubmed