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.

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...

We investigated the transfer characteristics and the gate-bias stability of amorphous indium-gallium-zinc oxide thin-film transistors when the channel layer was exposed to hydrogen, oxygen, air, or vacuum at room temperature during measurements. The threshold voltage and the drain current were changed by the ambient atmospheres. The threshold voltage shift (ΔVth) under gate-bias stress was faster in hydrogen than in oxygen and vacuum. It is suggested that hydrogen exposure degrades the gate-bias stress stability due to surface accumulation layer creation. The characteristic trapping times, τ, in H2, O2, air, and vacuum were 5×103, 1.5×104, 2×104, and 6.3×104 s, respectively.

The maintenance of telomere length is essential for the indefinite proliferation of cancer cells. This is most often achieved by the activation of telomerase; however, a substantial number of cancers lack detectable telomerase activity and are classified as using an alternative lengthening of telomeres (ALT) pathway. We showed recently that ALT cells have a high level of extrachromosomal telomeric circles (t circles) that may be a specific marker of the ALT phenotype. The mechanism underlying t circle production and the requirement of t circles in ALT remain unclear. Understanding the specific requirements of ALT is key to developing diagnostic tools and therapies that target this pathway and is critical for the treatment of cancers in which ALT is prevalent, including cancers of neuroepithelial and mesenchymal origin. In this study, we used short hairpin RNAs directed at either Xrcc3 or Nbs1, two proteins involved in the homologous recombination pathway, to determine the role of these proteins in t circle production and the requirement of t circles in maintaining the ALT pathway. We show that Xrcc3 and Nbs1 are indeed required for the production of t circles in human ALT. However, these cells continue to proliferate in the absence of t circles, suggesting that they are not required for the survival of ALT cells. [Cancer Res 2007;67(4):1513-9]

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The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA.

Background: Human excision repair removes UV photoproducts in 30-mers in vitro, but this has not been previously observed in vivo. Results: UV photoproducts are removed in vivo as 30-mers in complex with TFIIH both in general repair and in transcriptioncoupled repair. Conclusion: Primary products of excision repair have been isolated in vivo for the first time. Significance: The study provides novel insights into post-excision steps of human DNA repair.