Redox-sensitive alteration of replisome architecture safeguards genome integrity

Science ◽  
2017 ◽  
Vol 358 (6364) ◽  
pp. 797-802 ◽  
Author(s):  
Kumar Somyajit ◽  
Rajat Gupta ◽  
Hana Sedlackova ◽  
Kai John Neelsen ◽  
Fena Ochs ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Replication Fork ◽  
Genome Duplication ◽  
Genome Integrity ◽  
Oxygen Species ◽  
Oligomeric State ◽  
Replication Fork Progression ◽  
Metabolic Conditions ◽  
Low Levels ◽  
Deoxynucleotide Triphosphate

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)–generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.

scholarly journals Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
André Franz ◽  
Paul A. Pirson ◽  
Domenic Pilger ◽  
Swagata Halder ◽  
Divya Achuthankutty ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dynamic Process ◽  
Metabolic Pathways ◽  
Replication Fork ◽  
Genome Instability ◽  
Genome Integrity ◽  
Substrate Selection ◽  
Replication Fork Progression ◽  
Replication Factors ◽  
Spatiotemporal Control

Abstract The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

scholarly journals Basal CHK1 activity safeguards its stability to maintain intrinsic S-phase checkpoint functions

2019 ◽  
Vol 218 (9) ◽  
pp. 2865-2875 ◽  
Author(s):  
Jone Michelena ◽  
Marco Gatti ◽  
Federico Teloni ◽  
Ralph Imhof ◽  
Matthias Altmeyer
Keyword(s):  
Cell Proliferation ◽  
Steady State ◽  
Cell Survival ◽  
Replication Fork ◽  
S Phase ◽  
Genome Integrity ◽  
Replication Machinery ◽  
Replication Fork Progression ◽  
Steady State Activity ◽  
S Phase Checkpoint

The DNA replication machinery frequently encounters impediments that slow replication fork progression and threaten timely and error-free replication. The CHK1 protein kinase is essential to deal with replication stress (RS) and ensure genome integrity and cell survival, yet how basal levels and activity of CHK1 are maintained under physiological, unstressed conditions is not well understood. Here, we reveal that CHK1 stability is controlled by its steady-state activity during unchallenged cell proliferation. This autoactivatory mechanism, which depends on ATR and its coactivator ETAA1 and is tightly associated with CHK1 autophosphorylation at S296, counters CHK1 ubiquitylation and proteasomal degradation, thereby preventing attenuation of S-phase checkpoint functions and a compromised capacity to respond to RS. Based on these findings, we propose that steady-state CHK1 activity safeguards its stability to maintain intrinsic checkpoint functions and ensure genome integrity and cell survival.

scholarly journals The CMG helicase bypasses DNA protein cross-links to facilitate their repair

2018 ◽  
Author(s):  
Justin L. Sparks ◽  
Alan O. Gao ◽  
Markus Räschle ◽  
Nicolai B. Larsen ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Cross Link ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Cross Links ◽  
Leading Strand ◽  
Nucleoprotein Complexes ◽  
Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

scholarly journals CHK1 inhibition exacerbates replication stress induced by IGF blockade

Oncogene ◽  
2021 ◽  
Author(s):  
Xiaoning Wu ◽  
Elena Seraia ◽  
Stephanie B. Hatch ◽  
Xiao Wan ◽  
Daniel V. Ebner ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Replication Fork ◽  
Synthetic Lethality ◽  
Growth Factor Receptor ◽  
Replication Stress ◽  
Regulatory Subunit ◽  
Breast Cancer Cell Lines ◽  
Replication Fork Progression ◽  
Deoxynucleotide Triphosphate

AbstractWe recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

scholarly journals Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

2010 ◽  
Vol 191 (7) ◽  
pp. 1285-1297 ◽  
Author(s):  
Xin Quan Ge ◽  
J. Julian Blow
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Apparent Contrast ◽  
Origin Firing ◽  
Checkpoint Kinases ◽  
Replicative Stress ◽  
Replication Fork Progression ◽  
Replication Factory ◽  
Low Levels ◽  
Fork Stalling

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

scholarly journals Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage

2021 ◽  
Vol 8 ◽  
Author(s):  
Thomas A. Guilliam
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Replication Fork ◽  
Genome Stability ◽  
Genome Duplication ◽  
Damage Tolerance ◽  
Tolerance Mechanisms ◽  
Dna Damage Tolerance ◽  
Replication Fork Progression ◽  
The Impact

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

scholarly journals MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

2019 ◽  
Author(s):  
Sean G Rudd ◽  
Helge Gad ◽  
Nuno Amaral ◽  
Anna Hagenkort ◽  
Petra Groth ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Mitotic Cell ◽  
Mitotic Arrest ◽  
Oxygen Species ◽  
Mitotic Progression ◽  
Dna Polymerase Delta ◽  
Replication Fork Progression ◽  
In Cells

ABSTRACTReactive oxygen species (ROS) oxidise nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidised nucleotides by preventing incorporation via the oxidised purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidised nucleotides in cells or how oxidised nucleotides in DNA become toxic. We show replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), to an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with the MTH1 inhibitor (MTH1i) TH588. The resulting elevated genomic 8-oxodG correlates with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression is observed, but rather mitotic progression is impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevents accumulation of genomic 8-oxodG and reduces cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS build-up and oxidation of the nucleotide pool. Furthermore, we demonstrate delayed mitosis and increased mitotic cell death following TH588 treatment in cells expressing the error-prone Pol δ variant, which is not observed following treatments with anti-mitotic agents, thus linking incorporation of oxidised nucleotides and disturbed mitotic progression.

scholarly journals A novel multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress

2021 ◽  
Author(s):  
Wezley C. Griffin ◽  
David R. McKinzey ◽  
Kathleen N. Klinzing ◽  
Rithvik Baratam ◽  
Michael A. Trakselis
Keyword(s):  
Functional Role ◽  
Replication Fork ◽  
Active Role ◽  
Genome Integrity ◽  
Minichromosome Maintenance ◽  
Replication Fork Progression ◽  
Genomic Damage ◽  
High Sequence Homology ◽  
Fork Stalling ◽  
Functional Partitioning

AbstractThe minichromosome maintenance (MCM) 8/9 helicase is a AAA+ complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, an active role for MCM8/9 has remained elusive. We interrogated fork progression in cells lacking MCM8 or 9 and find there is a functional partitioning. Loss of MCM8 or 9 slows overall replication speed and increases markers of genomic damage and fork instability, further compounded upon treatment with hydroxyurea. MCM8/9 acts upstream and antagonizes the recruitment of BRCA1 in nontreated conditions. However, upon treatment with fork stalling agents, MCM9 recruits Rad51 to protect and remodel persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon excessive stalling, MCM8/9 directs additional stabilizers to protect forks from degradation. This evidence defines novel multifunctional roles for MCM8/9 in promoting normal replication fork progression and promoting genome integrity following stress.

scholarly journals MRNIP is a replication fork protection factor

2020 ◽  
Vol 6 (28) ◽  
pp. eaba5974 ◽  
Author(s):  
L. G. Bennett ◽  
A. M. Wilkie ◽  
E. Antonopoulou ◽  
I. Ceppi ◽  
A. Sanchez ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Replication Fork ◽  
Chromosome Instability ◽  
Genome Integrity ◽  
Replication Forks ◽  
Protection Factor ◽  
Replication Fork Progression ◽  
Dna Junctions ◽  
Genomic Regions ◽  
Stalled Replication Forks

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease–dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

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