The Rad4TopBP1 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner

Su-Jiun Lin,Izumi Miyabe,Christopher P Wardlaw,Thomas Caspari,Antony M Carr,Charly Chahwan,Takashi Morishita,Valerie Garcia,Ulrike Schmidt

doi:10.1371/journal.pgen.1002801

Su-Jiun Lin, Izumi Miyabe + Show 7 more

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https://doi.org/10.1371/journal.pgen.1002801

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Abstract

DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.

Highlights

The DNA damage checkpoint is an elaborate signal transduction pathway that monitors the integrity of the DNA, prevents cell cycle progression and promotes appropriate DNA metabolism [1] reviewed in [2]
We demonstrate that this domain is not required for initial checkpoint activation, but functions to amplify the checkpoint signal, likely when the presence of single-stranded DNA is limiting
Our data suggest that the function of the Rad4TopBP1 ATR-Activation Domain (AAD) is mediated by interactions between checkpoint proteins and phosphorylated histone H2A, which is itself promoted by Rad3ATR

Summary

Introduction

The DNA damage checkpoint is an elaborate signal transduction pathway that monitors the integrity of the DNA, prevents cell cycle progression and promotes appropriate DNA metabolism [1] reviewed in [2]. ATM detects DNA double strand breaks (DSBs) by interaction with the Mre11Rad50-Nbs repair complex, while ATR primarily senses single stranded-DNA (ss-DNA) through interactions with RPA. Both ATM and ATR are conserved in the model organisms S. pombe and S. cerevisiae. ATR-ATRIP recruitment to ssDNA-RPA is necessary for ‘‘basal’’ ATR activation, but is insufficient for full checkpoint activation: corecruitment of a second complex consisting of three PCNA-like proteins, Rad, Hus and Rad (known as the 9-1-1 clamp) is necessary. ATR-ATRIP recruitment to ssDNA-RPA is necessary for ‘‘basal’’ ATR activation, but is insufficient for full checkpoint activation: corecruitment of a second complex consisting of three PCNA-like proteins, Rad, Hus and Rad (known as the 9-1-1 clamp) is necessary. 9-1-1 is loaded in parallel to ATR recruitment at 59 ssDNA/dsDNA junctions by the checkpoint clamp loader Rad17RFC[2,3,4,5] [9,10,11]. (Figure 1A)

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