The Rad4TopBP1 ATR-activation domain functions in G1/S phase in a chromatin-dependent manner

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. © 2012 Lin et al.