Multiple ATR-Chk1 pathway proteins preferentially associate with checkpoint-inducing DNA substrates

Abstract

The ATR-Chk1 DNA damage checkpoint pathway is a critical regulator of the cellular response to DNA damage and replication stress in human cells. The variety of environmental, chemotherapeutic, and carcinogenic agents that activate this signal transduction pathway do so primarily through the formation of bulky adducts in DNA and subsequent effects on DNA replication fork progression. Because there are many protein-protein and protein-DNA interactions proposed to be involved in activation and/or maintenance of ATR-Chk1 signaling in vivo, we systematically analyzed the association of a number of ATR-Chk1 pathway proteins with relevant checkpoint-inducing DNA structures in vitro. These DNA substrates included single-stranded DNA, branched DNA, and bulky adduct-containing DNA. We found that many checkpoint proteins show a preference for single-stranded, branched, and bulky adduct-containing DNA in comparison to undamaged, double-stranded DNA. We additionally found that the association of checkpoint proteins with bulky DNA damage relative to undamaged DNA was strongly influenced by the ionic strength of the binding reaction. Interestingly, among the checkpoint proteins analyzed the checkpoint mediator proteins Tipin and Claspin showed the greatest differential affinity for checkpoint-inducing DNA structures. We conclude that the association and accumulation of multiple checkpoint proteins with DNA structures indicative of DNA damage and replication stress likely contribute to optimal ATR-Chk1 DNA damage checkpoint responses.

Figure 1. Purification of ATR-Chk1 pathway checkpoint proteins.

Recombinant forms of the indicated checkpoint proteins were separated by SDS-PAGE and visualized by coomassie blue staining. Proteins are categorized into (A) DNA damage sensors, (B) mediators, and (C) transducers/effectors.

Figure 2. RPA, FAAP24, and Chk1 preferentially bind ssDNA and branched DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to single-stranded (ss), double-stranded (ds), and branched, replication fork-like (fork) DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) RPA, (B) FAAP24, and (C) Chk1. Reactions were carried out as described in the Methods section. Experiments were repeated two to four times and the average binding values and standard deviations graphed. Input lane contains the lowest amount of protein used in each binding assay.

Figure 3. The DNA damage checkpoint sensors 9-1-1, ATRIP, and XPA preferentially bind branched DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to single-stranded (ss), double-stranded (ds), and branched, replication fork-like (fork) DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins were analyzed for binding to each DNA structure: (A) 9-1-1, (B) ATRIP, and (C) XPA. Reactions were carried out as described in the Methods section.

Figure 4. The DNA damage checkpoint mediators Claspin, Timeless, and Tipin preferentially bind branched DNA.

The association of each of the indicated purified proteins with no DNA (beads alone), single-stranded (ss), double-stranded (ds), and branched, replication fork-like (fork) DNA was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) Claspin, (B) Timeless-Tipin (Tim-Tipin) complex, (C) Timeless, and (D) Tipin. Reactions were carried out as described in the Methods section.

Figure 5. Association of TopBP1 and Cdc45 with DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to single-stranded (ss), double-stranded (ds), and branched, replication fork-like (fork) DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) TopBP1 and (B) Cdc45. Reactions were carried out as described in the Methods section.

Figure 6. FAAP24, Timeless, and Chk1 do not preferentially bind AAF-damaged DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to undamaged (−AAF) or N-acetoxy-2-acetylaminofluorene (AAF)-treated (+AAF) plasmid DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) FAAP24, (B) Timeless, and (C) Chk1. Reactions were carried out as described in the Methods section.

Figure 7. RPA, ATRIP, and XPA show a slight preference for AAF-damaged DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to undamaged (−AAF) or N-acetoxy-2-acetylaminofluorene (AAF)-treated (+AAF) plasmid DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) RPA, (B) ATRIP, and (C) XPA. Reactions were carried out as described in the Methods section.

Figure 8. Claspin and Tipin preferentially bind to AAF-damaged DNA.

The association of each of the indicated purified proteins with beads alone (No DNA) or to undamaged (−AAF) or N-acetoxy-2-acetylaminofluorene (AAF)-treated (+AAF) plasmid DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Each of the following proteins was analyzed for binding to each DNA structure: (A) Claspin and (B) Tipin. Reactions were carried out as described in the Methods section.

Figure 9. Effect of ionic strength on discrimination of damaged and undamaged DNA.

The association of each of TopBP1 with beads alone (No DNA) or to undamaged (−AAF) or AAF-damaged (+AAF) plasmid DNA bound to magnetic beads was analyzed by SDS-PAGE and immunoblotting. Binding reactions contained either (A) 100 mM NaCl, (B) 200 mM NaCl, or (C) 300 mM NaCl. Reactions were carried out as described in the Methods section.