An Essential Function for the ATR-Activation-Domain (AAD) of TopBP1 in Mouse Development and Cellular Senescence

Abstract

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

“…The 9-1-1 complex together with other proteins recruits TOPBP1, and TOPBP1 contains an ATR-activating domain with a critical residue, W1147, that binds ATR and stimulates its kinase activity (32). TOPBP1 appears to be the primary ATR activator under physiological mammalian cell replication, because homozygous null mutations or W1147 point mutations in mice result in early embryonic lethality at the blastocyst stage, phenocopying null mutations in Atr (11,33).…”

“…For anti-CD3/CD28 stimulation, CTV-labeled CD45.1 + Etaa1 +/+ and CD45.2 + Etaa1 ΔEx2/ΔEx2 splenocytes were mixed in a 50:50 ratio, and 5 × 10 5 cells/well were stimulated in 0.1-10 μg/mL soluble anti-CD3 (clone 500A2; BD Biosciences) with or without 2 μg/mL anti-CD28 (clone 37.15; BD Biosciences) in a flat-bottom 96-well plate. For in vitro P14 stimulation, 2 × 10 6 P14 total splenocytes per well were cultured in a flat-bottom 24-well plate with 10 ng/mL LCMV GP [33][34][35][36][37][38][39][40][41] peptide (Biomolecular Resource Facility of the John Curtin School of Medical Research, Australian National University) and 10 ng/mL recombinant human IL-2 (Peprotech). Daily cell counts were normalized via expression as fold change over the starting number of CD8 + Vα2 + P14 cells.…”

“…Flow cytometry staining with tetramers enabled enumeration of CD8 + T cells specific for two immunodominant epitopes of the virus, GP [33][34][35][36][37][38][39][40][41] and NP [396][397][398][399][400][401][402][403][404] , and CD4 + T cells specific for the immunodominant GP 66-77 virus epitope. Compared with wild-type controls, there were 10-fold fewer antigen-specific CD8 + and CD4 + T cells in LCMV-infected homozygotes for either the exon 2 deleted or the C166X null allele, although there was still a detectable effector T-cell response in both strains (Fig.…”

“…To track CD8 + T cells bearing a defined T-cell receptor (TCR) for virus antigen, we crossed the Etaa1 ΔEx2 allele to congenically marked (CD45.1 + ) P14 TCR transgenic C57BL/6 mice, which express a TCR specific for LCMV GP [33][34][35][36][37][38][39][40][41] antigen. Naïve CD8 + T cells expressing the P14 TCR developed and accumulated normally despite ETAA1 deficiency.…”

“…6C). To test in vitro proliferative capacity following antigen-specific stimulation, we stimulated Etaa1 +/+ or Etaa1-ΔEx2/ΔEx2 P14 CD8 + T cells with GP [33][34][35][36][37][38][39][40][41] peptide and IL-2 in vitro and analyzed them daily for 5 d. ETAA1-deficient P14 cells acquired the Ki67 cell cycle marker and increased in number comparably to their wild-type counterparts, and also exhibited a comparable percentage of viable cells at each time point (Fig. 6D), demonstrating that ETAA1 is dispensable for clonal expansion in vitro.…”