ATR activation is regulated by dimerization of ATR activating proteins

Vaughn Thada,David Cortez

doi:10.1016/j.jbc.2021.100455

Abstract

The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a “mini-ETAA1” protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuclei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization is important for optimal ATR signaling and genome stability.

Highlights

ataxia telangiectasia and Rad3-related (ATR) activation occurs in response to replication stress, and during normal DNA replication and in mitosis [3,4,5,6]
We previously identified the minimal DNA topoisomerase II binding protein 1 (TOPBP1) and ETAA1 activator of ATR kinase (ETAA1) activation domains (AADs) by determining which TOPBP1 and ETAA1 fragments were capable of activating ATR in an in vitro kinase assay [17]
These results indicate that a GST tag fused to the TOPBP1 and ETAA1 AADs enhances ATR activation without facilitating an interaction with the kinase substrate

Summary

Results

We previously identified the minimal TOPBP1 and ETAA1 AADs by determining which TOPBP1 and ETAA1 fragments were capable of activating ATR in an in vitro kinase assay [17]. Even at a threefold higher concentration, ATR activation by untagged TOPBP1 or ETAA1 AADs is still less than activation by the corresponding GST-AADs (compare lanes 2 and 5 and 6 and 9) (Fig. 1A). Compared to GST alone, GSTTOPBP1 AAD strongly stimulates ATR kinase activity towards the untagged substrate, while the untagged TOPBP1 AAD does not (Fig. 1B). We determined how another tag that does not dimerize, MBP, affects ATR activation by the AADs. In contrast to the GST-TOPBP1 AAD, the MBPTOPBP1 AAD induces minimal, if any, ATR activation toward the untagged substrate (Fig. 1B). We observed the same result with GST-tagged, MBP-tagged, and untagged ETAA1 AADs (Fig. 1C) Together, these results indicate that a GST tag fused to the TOPBP1 and ETAA1 AADs enhances ATR activation without facilitating an interaction with the kinase substrate. Dimerization of proteins fused to FKBP F36V (hereafter referred to as FK) occurs upon incubation with the rapamycin analog AP20187

A FKBP F36V

C ETAA1 2-305 2 AAD 305

Discussion

Experimental procedures