Journal Article

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

Lindsey-Boltz, Laura A., Mason, Aaron C., Kemp, Michael, Choi, Jun-Hyuk, Wold, Marc S., Sancar, Aziz

2010

Overview

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Adaptor Proteins, Signal Transducing - isolation & purification / Adaptor Proteins, Signal Transducing - metabolism / Ataxia Telangiectasia Mutated Proteins / Binding sites / Biological Sciences / Carrier Proteins - metabolism / Cell Cycle Proteins - isolation & purification / Cell Cycle Proteins - metabolism / Cell free system / Checkpoint Kinase 1 / Deoxyribonucleic acid / DNA / DNA damage / DNA repair / DNA Repair Enzymes - isolation & purification / DNA Repair Enzymes - metabolism / DNA replication / DNA, Single-Stranded - chemistry / DNA, Single-Stranded - metabolism / DNA-Binding Proteins - metabolism / Enzyme Activation / Genetic mutation / HeLa Cells / Humans / Immunoblotting / Kinases / mutants / Mutation / Nuclear Proteins - metabolism / Nucleic Acid Conformation / Phosphorylation / Protein Binding / Protein Kinases - metabolism / Protein-Serine-Threonine Kinases - isolation & purification / Protein-Serine-Threonine Kinases - metabolism / Proteins / Replication Protein A - metabolism / Signal Transduction / single-stranded DNA / Yeasts