Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks

Bekker‐Jensen, Simon; Lukas, Claudia; Kitagawa, Risa; Melander, Fredrik; Kastan, Michael B.; Bártek, Jiří; Lukáš, Jiří

doi:10.1083/jcb.200510130

Cited by 569 publications

(621 citation statements)

References 49 publications

(82 reference statements)

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“…Each of these signalling cascades involves several unique and overlapping factors -classified as sensors, mediators, transducers and effectors -that ultimately lead to the spatio-temporal assembly of multi-protein complexes at the site of damage 1,2 . The functional importance of checkpoints in maintaining genome stability is highlighted by their conservation throughout eukaryotes.…”

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confidence: 99%

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

et al. 2007

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Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.The DNA damage response (DDR) is a complex process involving the orchestration of highly specialized cell-cycle checkpoints that need to be rapidly activated following the detection of damaged DNA. Each of these signalling cascades involves several unique and overlapping factors -classified as sensors, mediators, transducers and effectors -that ultimately lead to the spatio-temporal assembly of multi-protein complexes at the site of damage 1,2 . The functional importance of checkpoints in maintaining genome stability is highlighted by their conservation throughout eukaryotes. As such, defective checkpoint pathways in human cells leads to the accumulation of aberrant DNA and an increased risk of cancer progression, evident from the many human disease syndromes that result from defects in checkpoint factors 3,4 . Therefore, it is important to understand these complex mechanisms at the molecular level to further our knowledge of cancer progression and treatment.Checkpoints operate at the G1-S, S and G2-M boundaries of the cell cycle and are controlled by the ATM-Chk2 and ATR-Chk1 pathways. The S-phase checkpoint, which is under the control of the ATR-Chk1 pathway, has an essential role in maintaining replication-fork integrity during normal S-phase by preventing the collapse of stalled replication forks. The S-phase checkpoint also coordinates cell-cycle arrest and subsequent DNA-repair events when the replication fork encounters DNA damage 5,3,6 . Activation and recruitment of the Fanconi anaemia and homologous recombination-repair pathways to sites of replication stress requires an intact S-phase checkpoint 7 .Recent work in mouse models has highlighted a surprising, yet highly important link between biological clock proteins and the DDR 8,9 . It has been shown that two clock proteins, Per1 and Prd4, have integral roles...

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confidence: 99%

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

et al. 2007

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“…It is difficult to test the former, since normal numbers of gH2AX foci are observed even in the absence of ATM (McManus and Hendzel, 2005). In addition, while further activation involves MRN, it is also evident that MDC1 plays an important role in amplifying ATM-dependent DNA-damage signalling (Bekker-Jensen et al, 2006;Lou et al, 2006). This may be a later event with MDC1, providing a platform to stabilize the complex and sustain ATM signalling.…”

Section: Perspectivementioning

confidence: 99%

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

2007

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The recognition and repair of DNA double-strand breaks (DSBs) is a complex process that draws upon a multitude of proteins. This is not surprising since this is a lethal lesion if left unrepaired and also contributes to genome instability and the consequential risk of cancer and other pathologies. Some of the key proteins that recognize these breaks in DNA are mutated in distinct genetic disorders that predispose to agent sensitivity, genome instability, cancer predisposition and/or neurodegeneration. These include members of the Mre11 complex (Mre11/Rad50/ Nbs1) and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human genetic disorder A-T. The mre11 (MRN) complex appears to be the major sensor of the breaks and subsequently recruits ATM where it is activated to phosphorylate in turn members of that complex and a variety of other proteins involved in cellcycle control and DNA repair. The MRN complex is also upstream of ATM and ATR (A-T-mutated and rad3-related) protein in responding to agents that block DNA replication. To date, more than 30 ATM-dependent substrates have been identified in multiple pathways that maintain genome stability and reduce the risk of disease. We focus here on the relationship between ATM and the MRN complex in recognizing and responding to DNA DSBs.

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“…The radiation employed for this purpose ranges from X-rays [6] to charged particles [7] to laser emission at various wavelengths [8][9][10][11]. For proteins that are attracted to DNA lesions, as e.g.…”

Section: Biophotonicsmentioning

confidence: 99%

“…The laser beam is then scanned along a trajectory of choice or directed to a subnuclear structure of interest. These recruitment analyses are very useful to establish the temporal order of binding of different DNA repair factors at the damage by comparing their recruitment kinetics [9]. They are not per se mobility measurements and thus do not assess how DNA damage alters the dynamics of an individual protein of interest.…”

Section: Biophotonicsmentioning

confidence: 99%

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

Tomas

Blumhardt

Deutzmann

et al. 2012

Journal of Biophotonics

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Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks

Cited by 569 publications

References 49 publications

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

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