# Mechanistic analysis of ATR signaling

> **NIH NIH R01** · UNIVERSITY OF SOUTHERN CALIFORNIA · 2020 · $330,000

## Abstract

Project Summary/Abstract
The ATR protein kinase sits atop a complex signaling cascade that is activated by DNA replication and hyper-
activated by replication stress or DNA double-strand breaks. Activation of ATR by genotoxic stress is essential
for the ability of cells to survive stress and represents a barrier to transformation of normal cells to a
pathological condition that promotes tumorigenesis. The downstream effectors and consequences of ATR
signaling are now being understood, however early events in signaling, such as the initial activation of ATR
kinase at sites of gentoxic stress, are still poorly understood. This proposal focuses on the biochemical
mechanism for ATR activation at sites of DNA damage. This proposal features the TOPBP1 protein, which
physically interacts with ATR at sites of DNA damage and is required for ATR activation during the replication
stress and DNA damage responses. TOPBP1 is a BRCT repeat containing protein that likely acts as a scaffold to
link checkpoint proteins together into active signaling centers. In this proposal we combine biochemical
studies using purified factors, functional studies in Xenopus egg extracts, and in vivo studies in cultured cells
to mount an in-depth exploration of how TOPBP1 activates ATR at sites of DNA damage. Recent studies on the
ETAA1 protein have shown that TOPBP1 is not alone in its ability to activate ATR, and our preliminary studies
suggest that a third and possibly more ATR activators are present in human cells. In this proposal we will also
study these new activators, with the long-term goal of building a comprehensive, systems-level view of how
ATR signaling is initiated.
The work is divided into three Aims. In Aim 1, we examine how active ATR signaling centers form at sites of
damage. The recruitment mechanism is complex, and likely involves multiple protein-protein interactions
between TOPBP1 and its binding partners. Recent work from our group has shown that soluble TOPBP1 is held
in an auto-inhibitory conformation and thus an additional goal of Aim 1 is to determine how this auto-
inhibition is resolved at sites of damage. In Aim 2 we examine how ATR may use a negative feedback loop to
regulate assembly of additional signaling centers at sites of damage. We will also explore the possibility that an
ATR signaling center is mobile on DNA. In the final Aim we will define a rule-book for how different ATR
activators are utilized, how their functions are related (or not), and how their activities are integrated during an
ATR response.

## Key facts

- **NIH application ID:** 10004100
- **Project number:** 5R01GM122887-04
- **Recipient organization:** UNIVERSITY OF SOUTHERN CALIFORNIA
- **Principal Investigator:** Matthew Michael
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $330,000
- **Award type:** 5
- **Project period:** 2017-09-08 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10004100

## Citation

> US National Institutes of Health, RePORTER application 10004100, Mechanistic analysis of ATR signaling (5R01GM122887-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10004100. Licensed CC0.

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