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Checking on DNA damage in S phase

Key Points

  • To protect the integrity of their genome, eukaryotic cells possess evolutionarily conserved surveillance mechanisms that are known as checkpoints, which constantly monitor the status and quality of chromosomal DNA and delay cell-cycle progression in response to replication stress or diverse types of DNA damage.

  • The genetically most vulnerable period of the cell cycle, the DNA-synthesis (S) phase, is protected against various genotoxic stresses by three checkpoints. These are the DNA-damage-induced, replication-independent, intra-S-phase checkpoint (the 'intra-S-phase checkpoint'), which is the main topic of this review; the replication-dependent S-phase checkpoint (the 'replication checkpoint'); and the replication-dependent S–M checkpoint (the 'S–M checkpoint'), which prevents mitotic entry when DNA is incompletely replicated.

  • Recent discoveries reveal the distinct molecular mechanisms that activate the two most proximal checkpoint-signalling kinases ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and RAD3 related (ATR), respectively. Furthermore, new evidence sheds light on the identity and function of candidate DNA-damage sensors, such as the NBS1–MRE11–RAD50 (MRN) complex (where NBS1 stands for Nijmegen-breakage-syndrome-1 and MRE11 stands for meiotic-recombination protein-11), as well as other components of the checkpoint-signalling cascades including the effectors. In response to DNA double-strand breaks (DSBs), the activated intra-S-phase checkpoint inhibits DNA replication through the combined action of the ATM/ATR–CHK2/CHK1–CDC25A–CDK2 and the ATM–NBS1–FANCD2/SMC1 effector pathways (where CHK stands for checkpoint kinase; CDK stands for cyclin-dependent kinase; FANCD2 stands for Fanconi anaemia complementation group D2; and SMC1 stands for structural maintenance of chromosomes-1).

  • A recently identified class of proximal checkpoint regulators, termed mediators, include p53-binding protein-1 (53BP1), mediator of DNA-damage checkpoint-1 (MDC1) and breast cancer susceptibility protein-1 (BRCA1) for the ATM-mediated responses, and claspin for ATR. They seem to serve as molecular match-makers that are critical for the timely and coordinated activation and maintenance of the checkpoint responses.

  • Live-cell, real-time imaging of early molecular events that are set in motion after formation of DSBs has provided valuable information about the spatio-temporal orchestration of the DSB response. This includes an unexpectedly dynamic behaviour of the checkpoint proteins that are involved in the recognition, processing, signalling and repair of the DNA lesions.

  • Defects in the genes that encode products that participate in the intra-S-phase checkpoint disable the checkpoint response, and thereby cause unscheduled DNA synthesis in irradiated cells (known as radioresistant DNA synthesis; RDS). Such checkpoint defects cause predisposition to a range of life-threatening human diseases including cancer.

Abstract

The precise replication of the genome and the continuous surveillance of its integrity are essential for survival and the avoidance of various diseases. Cells respond to DNA damage by activating a complex network of the so-called checkpoint pathways to delay their cell-cycle progression and repair the defects. In this review we integrate findings on the emerging mechanisms of activation, the signalling pathways and the spatio-temporal organization of the intra-S-phase DNA-damage checkpoint and its impact on the cell-cycle machinery, and discuss its biological significance.

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Figure 1: Distinct modes of DSB-induced activation of the ATM and ATR kinases.
Figure 2: Checkpoint-induced degradation of the CDC25A phosphatase.
Figure 3: Phosphorylation-mediated control of CDC25A protein turnover.
Figure 4: DSB-induced phosphorylation of SMC1.
Figure 5: Spatio-temporal regulation of the intra-S-phase checkpoint.

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Acknowledgements

The authors' research is supported by the Danish Cancer Society and the European Union. The authors apologize to colleagues whose work could only be cited indirectly due to space limitations.

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Correspondence to Jiri Bartek.

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Supplementary information

Supplementary information S1

Supplementary information S1 (movie) |A real-time monitoring of NBS1 and CHK2 redistribution in response to spatially restricted DSBs. Human osteosarcoma cells (U-2-OS) that express low levels of green fluorescent protein (GFP)-tagged checkpoint kinase-2 (CHK2) and yellow fluorescent protein (YFP)-tagged Nijmegen-breakage-syndrome-1 (NBS1) were mixed and micro-irradiated within the linear subnuclear compartments by a focused laser beam (λ=337 nm). When combined with the presensitization of cells with halogenated thymidine analogues, this treatment results in the generation of DNA double-strand breaks (DSBs) or single-strand breaks specifically in the laser-exposed nuclear areas1. Whereas the NBS1 protein underwent a rapid (on the order of seconds) recruitment to, and accumulation around, the damaged nuclear areas, CHK2 remained highly mobile and disseminated throughout the nucleoplasm. Parallel immunostaining with phospho-specific antibodies confirmed that after a very transient 'collision' with the freshly generated DSBs, CHK2 was distributed throughout the nucleus in its activated (ATM–phosphorylated) form2. Red arrows indicate the laser tracks; the entire movie spans the first 10 minutes after micro-irradiation. (MP4 93 kb)

1. Limoli, C. L. & Ward, J. F. A new method for introducing double-strand breaks into cellular DNA. Radiat. Res. 134, 160–169 (1993). 2. Lukas, C., Falck, J., Bartkova, J., Bartek, J. & Lukas, J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nature Cell Biol. 5, 255–260 (2003).

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DATABASES

OMIM

Ataxia-telangiectasia

ATR

Seckel syndrome

Saccharomyces genome database

TEL1

XRS2

Swiss-Prot

ATM

ATRIP

53BP1

BRCA1

CDC25A

CHK1

CHK2

claspin

H2AX

MRE11

NBS1

PP5

RAD50

SMC1

β-TrCP

Glossary

INTRA-S-PHASE CHECKPOINT

Replication-independent mechanism that reduces the rate of DNA synthesis in response to DNA double-strand breaks.

REPLICATION CHECKPOINT

A mechanism that blocks S-phase progression in cells that are experiencing stalled replication forks.

S–M CHECKPOINT

A cellular mechanism that prevents mitotic onset in cells with incompletely replicated DNA.

RADIOMIMETIC DRUG

A chemical that has similar effects as ionizing radiation, causing the generation of DNA double-strand breaks.

REPLICON

A unit of chromosomal DNA that is replicated by initiation at a single replication origin.

REPLICATION ORIGIN

A site on chromosomal DNA where replicative DNA synthesis is initiated. Replication origins in human cells are divided into more than 1,000 so-called replication zones, each of which is activated at a characteristic time during S phase.

RADIORESISTANT DNA SYNTHESIS

(RDS). The inability to slow down DNA replication in response to ionizing radiation and/or radiomimetic drugs.

INTERMOLECULAR AUTOPHOSPHORYLATION

The exchange of phosphate groups between two identical protein kinases.

MRN NUCLEASE COMPLEX

A protein complex that comprises the MRE11, RAD50 and NBS1 subunits, and that is involved in the recognition, processing, signalling and repair of DNA double-strand breaks. The nuclease activity is associated with the MRE11 subunit.

9-1-1 COMPLEX

A heterotrimeric protein complex that comprises the RAD9, RAD1 and HUS1 subunits, and that forms a 'doughnut-shaped' sliding clamp around DNA after damage and/or replication-fork stalling. Its loading onto DNA requires RAD17 (the so-called 'clamp loader' protein).

REPLICATION PROTEIN A

(RPA). A protein that binds and protects single-stranded DNA.

FHA DOMAIN

The forkhead-associated domain is a phospho-threonine-binding motif that was first identified in forkhead transcription factors and, subsequently, in other classes of proteins — including those that are involved in checkpoint signalling.

BRCT DOMAIN

An evolutionarily conserved phospho-serine/threonine-interaction motif that was first identified in the carboxy-terminal part of BRCA1 and, subsequently, in several other checkpoint mediators.

COHESIN

A proteinaceous ring that consists of four subunits (SMC1, SMC3, SCC1, SCC3) and that forms around the replicated DNA strands. Cohesin prevents premature separation of the nascent sister chromatids and some of its components (SMC1) participate in DNA recombination.

F-BOX PROTEINS

A family of proteins that function as the substrate-targeting protein subunits of the SCF (SKP1–CUL1–F-box protein) ubiquitin-ligase complex.

PHOSPHODEGRON MOTIF

A protein domain that, on phosphorylation, serves as a docking site for a specific ubiquitin ligase.

PSORALEN

A compound that sensitizes mammalian cells to ultraviolet radiation.

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Bartek, J., Lukas, C. & Lukas, J. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5, 792–804 (2004). https://doi.org/10.1038/nrm1493

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