Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

Zou, Lee

doi:10.1101/gad.1550307

Cited by 116 publications

(102 citation statements)

References 57 publications

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“…However, the system in its present form has certain limitations. First, there are extensive data that primer/template structures, in which the singlestranded region is covered by RPA, generated by replication blocks or by nucleotide excision repair are strong signals for checkpoint activation (22). However, those data and our in vitro findings in this paper and in vivo data published previously (24) are not necessarily mutually exclusive because it is quite likely that more than one type of DNA/protein structure may constitute a signal for checkpoint activation.…”

Section: Discussionmentioning

confidence: 64%

“…However, those data and our in vitro findings in this paper and in vivo data published previously (24) are not necessarily mutually exclusive because it is quite likely that more than one type of DNA/protein structure may constitute a signal for checkpoint activation. Second, in vivo data indicate that in addition to TopBP1, several other proteins, such as Rad17-RFC, the 9-1-1 complex, Claspin, and Timeless are required for phosphorylation of Chk1 by ATR (3,22). Indeed, a recent study indicates that the Rad9 subunit of the 9-1-1 complex plays a role in recruitment of TopBP1 to chromatin and thus facilitates ATR activation by this mechanism (31).…”

Section: Discussionmentioning

confidence: 99%

“…However, the stimulatory effect was not specific to deca-primed DNA, because we observed essentially the same levels of stimulation of the TopBP1-dependent ATR kinase activity by both single-stranded (lanes 3-5) and doublestranded (lanes 9-11) plasmid DNAs. Because a number of studies have indicated that replication protein A (RPA)-covered single-stranded DNA is a common, if not universal, signal for ATR-mediated checkpoint activation (21,22), we tested the effect of RPA on the reaction by using both deca-primed and double-stranded DNA. We observed only a slight stimulation upon the addition of RPA with both types of DNA (SI Fig.…”

Section: Effects Of Various Dna Substrates On Topbp1-dependent Atr Kimentioning

confidence: 99%

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

Choi

Lindsey-Boltz

Sancar

2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Effects Of Various Dna Substrates On Topbp1-dependent Atr Kimentioning

confidence: 99%

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

Choi

Lindsey-Boltz

Sancar

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Since accumulation of ss DNA can trigger DDR by ATR activation [33,34], we next examined whether the excessive ss telomeric DNA in Stn1-KD cells triggered ATR activation by analyzing phosphorylation of its downstream target Chk1. Chk1 phosphorylation was nearly undetectable immediately following Stn1 KD ( Figure 3E, PD39).…”

Section: Deficiency Of Stn1 Induces Rapid Telomere Shortening Early mentioning

confidence: 99%

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

2012

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Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.

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“…First, a telomere with its 3′ G-strand overhang provides the classic structure (a 5′ double-stranded/single-stranded DNA (ds/ssDNA) junction) for ATR activation 19 . Second, Rpa (replication protein A), a key protein in ATR recruitment, is known to bind to telomeres during S phase 12 and hence is likely to protect the overhangs in late S/G2 in cells that lack functional Pot1.…”

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confidence: 99%

Pot1 and cell cycle progression cooperate in telomere length regulation

Churikov

Price

2007

Nat Struct Mol Biol

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Removal of the vertebrate telomere protein Pot1 results in a DNA damage response and cell cycle arrest. Here we show that loss of chicken Pot1 causes Chk1 activation, and inhibition of Chk1 signaling prevents the cell cycle arrest. However, arrest still occurs after disruption of ATM, which encodes another DNA damage response protein. These results indicate that Pot1 is required to prevent a telomere checkpoint mediated by another such protein, ATR, that is most likely triggered by the G-overhang. We also show that removal of Pot1 causes exceptionally rapid telomere growth upon arrest in late S/G2 of the cell cycle. However, release of the arrest slows both telomere growth and G-overhang elongation. Thus, Pot1 seems to regulate telomere length and G-overhang processing both through direct interaction with the telomere and by preventing a late S/G2 delay in the cell cycle. Our results reveal that cell cycle progression is an important component of telomere length regulation.Telomeres are essential for genome stability, because they prevent chromosome ends from being ligated together by the repair pathways that act on DNA double-strand breaks 1,2 . The telomeric DNA is shielded from double-strand break repair activities by specialized telomere proteins that bind along the duplex region of the telomere and to the overhang on the 3′ G-rich strand. In vertebrate cells the telomeric duplex is packaged by a complex of six proteins (Trf1, Trf2, Rap1, Tin2, Tpp1 and Pot1) termed shelterin 1 . Two of the proteins, Pot1 and Tpp1, also bind to the G-strand overhang. Pot1 is a specialized G-strand binding protein, whereas Tpp1 interacts with both telomerase and other components of the shelterin complex 3,4 .

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Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

Cited by 116 publications

References 57 publications

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

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

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

Pot1 and cell cycle progression cooperate in telomere length regulation

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