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

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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^TopBP1 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^TopBP1 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 Crb2^53BP1. Consistent with a model where Rad4^TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4^TopBP1 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^TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3^ATR 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.

Vyšlo v časopise: The Rad4 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner. PLoS Genet 8(6): e32767. doi:10.1371/journal.pgen.1002801
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002801

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