Reconstitution of a human ATR-mediated checkpoint response to damaged DNA

Abstract

The DNA damage checkpoint response delays cell cycle progression upon DNA damage and prevents genomic instability. Genetic analysis has identified sensor, mediator, signal transducer, and effector components of this global signal transduction pathway. Here we describe an in vitro system with purified human checkpoint proteins that recapitulates key elements of the DNA damage checkpoint. We show that the damage sensor ATR in the presence of topoisomerase II binding protein 1 (TopBP1) mediator/adaptor protein phosphorylates the Chk1 signal-transducing kinase in a reaction that is strongly dependent on the presence of DNA containing bulky base lesions. The dependence on damaged DNA requires DNA binding by TopBP1, and, indeed, TopBP1 shows preferential binding to damaged DNA. This in vitro system provides a useful platform for mechanistic studies of the human DNA damage checkpoint response.Chk1 kinase ͉ damage recognition ͉ signal transduction ͉ topoisomerase II binding protein 1 M ost of our knowledge about the DNA damage checkpoint response is based on genetic data from model organisms, including budding and fission yeasts and humans and the Xenopus egg extract system. These studies have identified damage sensors, mediators, signal transducers, and effectors as key components of this signal-transduction pathway (1-3). The phosphatidylinositol 3-kinase-related protein kinase (PIKK) family members have been shown to be key DNA damage sensors and signal transducers in the checkpoint response. Of these, ATM is mainly responsible for initiation of the checkpoint response elicited by double-strand breaks caused by ionizing radiation or radiomimetic agents. Some semidefined systems for the ATM-mediated checkpoint response have been described recently (4-8). Another PIKK family member, ATR, initiates the DNA damage checkpoint response caused by UV radiation and UV-mimetic agents that produce base damage such as N-acetoxy-2-acetylaminofluorene (N-Aco-AAF). Although this important signal-transduction pathway has been characterized in some detail by using Xenopus egg extracts (9-15) and in human cell-free systems (16, 17), only recently have partial in vitro systems been developed with a subset of either Xenopus (14, 15) or yeast (18) checkpoint proteins. Currently, there is no well defined system for ATR-mediated DNA damage checkpoint response in humans. Recently, it has been shown that the multifunctional XtopBP1 protein, which is known to be required for the ATR-mediated checkpoint (19), activates the ATR kinase on downstream targets, in particular the Chk1 signaltransducing kinase, in the absence of DNA or any other checkpoint protein except the ATR-interacting protein (ATRIP) (14). Here we describe a human in vitro system in which ATR phosphorylates Chk1 kinase in a reaction that depends on topoisomerase II binding protein 1 (TopBP1) and is strongly stimulated by DNA containing bulky DNA adducts. We believe this is a useful system for the ultimate development of an in vitro human checkpoint response dependent on all che...

Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

“…in the presence of damaged DNA (9). In the current report, we have bypassed the requirement for damaged DNA both in vitro and in vivo by fusing TopBP1 to LacR and using DNA containing LacR binding sites.…”

“…For the ATR pathway, there are many potential mechanisms for achieving stable chromatin association of the checkpoint proteins, such as by RPA-coated ssDNA, which, through interactions with ATRIP (27) and the checkpoint mediator protein Tipin (28), bring ATR and Claspin into close proximity to one another to facilitate Chk1 phosphorylation. Similarly, interactions between ATR and DNA repair factors (29 -32) as well as direct binding of checkpoint proteins to DNA structures such as branched DNA (23) or DNA-containing bulky adducts (9,33), may also facilitate activation of the ATR pathway.…”

“…Purification of Checkpoint Proteins-Native ATR-ATRIP, GST-TopBP1-His, RPA, and His-Chk1 kinase-dead (HisChk1-kd) were all purified as described previously (8,9,15). LacR-TopBP1 and LacR-Claspin-C WT and 3A were expressed in 293 FlpIn T-REX cells as described in the manufacturer's directions (Invitrogen), and purified with anti-FLAG-agarose (Sigma) as described previously (16).…”

“…We have previously described an in vitro system that recapitulates ATR phosphorylation of Chk1 dependent on RPA-coated ssDNA and TopBP1 (8). However, RPA is not required for maximal ATR kinase activity in this system when the ssDNA is replaced with DNA containing bulky DNA base adducts (9,10). In fact, we found that TopBP1 binds directly to damaged DNA and that the DNA binding activity of TopBP1 is required to confer damaged DNA-dependent activation of ATR.…”

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

“…The DNA was purified on a Qiagen columnandthentreatedwith300MN-acetoxy-2-acetylaminofluorene (AAAF) or ethanol (for unmodified DNA) for 3 h at room temperature in the dark. The AAAF treatment protocol yields ϳ1 AAAF-guanine adduct every 200 -250 bp of DNA, resulting in 10 -14 lesions per plasmid (42). After ether extraction to remove the unincorporated AAAF, the DNA was precipitated in ethanol, dried, and resuspended in TE (10 mM Tris acetate, pH 7.5, 1 mM EDTA).…”