The Rad4 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner

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 Rad4 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 Rad4 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 Crb2. Consistent with a model where Rad4 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3 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. Citation: Lin S-J, Wardlaw CP, Morishita T, Miyabe I, Chahwan C, et al. (2012) The Rad4 ATR-Activation Domain Functions in G1/S Phase in a ChromatinDependent Manner. PLoS Genet 8(6): e1002801. doi:10.1371/journal.pgen.1002801 Editor: Sue Jinks-Robertson, Duke University, United States of America Received March 5, 2012; Accepted May 17, 2012; Published June 28, 2012 Copyright: ! 2012 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by CRUK grant C5514/A10722 (www.cancerresearchuk.org/) and MRC grant G0600233 (www.mrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work.