scholarly journals Break-Induced Replication Repair of Damaged Forks Induces Genomic Duplications in Human Cells

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 88-91 ◽  
Author(s):  
Lorenzo Costantino ◽  
Sotirios K. Sotiriou ◽  
Juha K. Rantala ◽  
Simon Magin ◽  
Emil Mladenov ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Replication Stress ◽  
Replication Forks ◽  
Dna Polymerase Delta ◽  
Dna Double Strand Breaks ◽  
Dna Replication Stress ◽  
Recombination Pathway ◽  
Break Induced Replication

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

scholarly journals The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

2017 ◽  
Author(s):  
Naomi Coulton ◽  
Thomas Caspari
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Stress ◽  
Genetic Alterations ◽  
Activation Loop ◽  
Replication Forks ◽  
Dna Polymerase Delta ◽  
Dna Replication Stress ◽  
Summary Statement ◽  
Chk1 Kinase

AbstractWhy the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.Summary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

scholarly journals Physiological and Pathological Roles of RAD52 at DNA Replication Forks

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 402 ◽  
Author(s):  
Eva Malacaria ◽  
Masayoshi Honda ◽  
Annapaola Franchitto ◽  
Maria Spies ◽  
Pietro Pichierri
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
Molecular Mechanisms ◽  
Full Range ◽  
Therapeutic Targets ◽  
Replication Stress ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Rad52 Protein ◽  
New Biomarkers

Understanding basic molecular mechanisms underlying the biology of cancer cells is of outmost importance for identification of novel therapeutic targets and biomarkers for patient stratification and better therapy selection. One of these mechanisms, the response to replication stress, fuels cancer genomic instability. It is also an Achille’s heel of cancer. Thus, identification of pathways used by the cancer cells to respond to replication-stress may assist in the identification of new biomarkers and discovery of new therapeutic targets. Alternative mechanisms that act at perturbed DNA replication forks and involve fork degradation by nucleases emerged as crucial for sensitivity of cancer cells to chemotherapeutics agents inducing replication stress. Despite its important role in homologous recombination and recombinational repair of DNA double strand breaks in lower eukaryotes, RAD52 protein has been considered dispensable in human cells and the full range of its cellular functions remained unclear. Very recently, however, human RAD52 emerged as an important player in multiple aspects of replication fork metabolism under physiological and pathological conditions. In this review, we describe recent advances on RAD52’s key functions at stalled or collapsed DNA replication forks, in particular, the unexpected role of RAD52 as a gatekeeper, which prevents unscheduled processing of DNA. Last, we will discuss how these functions can be exploited using specific inhibitors in targeted therapy or for an informed therapy selection.

scholarly journals RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

2021 ◽  
Vol 12 ◽  
Author(s):  
Nathan Ellis ◽  
Jianmei Zhu ◽  
Mary K Yagle ◽  
Wei-Chih Yang ◽  
Jing Huang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Replication Stress ◽  
Dna Helicase ◽  
Replication Forks ◽  
Replication Origins ◽  
Dna Double Strand Breaks ◽  
Response Factors ◽  
Fork Stalling ◽  
In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

scholarly journals The evolutionary plasticity of chromosome metabolism allows adaptation to constitutive DNA replication stress

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marco Fumasoni ◽  
Andrew W Murray
Keyword(s):  
Dna Replication ◽  
Natural Populations ◽  
Replication Stress ◽  
Replication Forks ◽  
Yeast Saccharomyces Cerevisiae ◽  
Evolutionary Trajectory ◽  
Biological Features ◽  
Chromatid Cohesion ◽  
Conserved Genes ◽  
Dna Replication Stress

Many biological features are conserved and thus considered to be resistant to evolutionary change. While rapid genetic adaptation following the removal of conserved genes has been observed, we often lack a mechanistic understanding of how adaptation happens. We used the budding yeast, Saccharomyces cerevisiae, to investigate the evolutionary plasticity of chromosome metabolism, a network of evolutionary conserved modules. We experimentally evolved cells constitutively experiencing DNA replication stress caused by the absence of Ctf4, a protein that coordinates the enzymatic activities at replication forks. Parallel populations adapted to replication stress, over 1000 generations, by acquiring multiple, concerted mutations. These mutations altered conserved features of two chromosome metabolism modules, DNA replication and sister chromatid cohesion, and inactivated a third, the DNA damage checkpoint. The selected mutations define a functionally reproducible evolutionary trajectory. We suggest that the evolutionary plasticity of chromosome metabolism has implications for genome evolution in natural populations and cancer.

scholarly journals Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

2020 ◽  
Author(s):  
Zainab Tayeh ◽  
Kim Stegmann ◽  
Antonia Kleeberg ◽  
Mascha Friedrich ◽  
Josephine Ann Mun Yee Choo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Stress ◽  
List Type ◽  
Replication Forks ◽  
Loop Formation ◽  
Animal Cells ◽  
Replication Fork Progression ◽  
Primordial Dwarfism ◽  
Dna Replication Stress

AbstractCentrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication.HighlightsCentrosome defects cause replication stress independent of mitosis.MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation.Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation.Graphical abstract

scholarly journals Smarcal1 and Zranb3 Protect Replication Forks from Myc-Induced DNA Replication Stress

2019 ◽  
Vol 79 (7) ◽  
pp. 1612-1623 ◽  
Author(s):  
Matthew V. Puccetti ◽  
Clare M. Adams ◽  
Saul Kushinsky ◽  
Christine M. Eischen
Keyword(s):  
Dna Replication ◽  
Replication Stress ◽  
Replication Forks ◽  
Dna Replication Stress

scholarly journals Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

2021 ◽  
Author(s):  
Sandhya Balasubramanian ◽  
Matteo Andreani ◽  
Júlia Goncalves Andrade ◽  
Tannishtha Saha ◽  
Javier Garzón ◽  
...  
Keyword(s):  
Replication Stress ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Protective Function ◽  
Multifunctional Protein ◽  
Strand Breaks ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region ◽  
Dna Replication Stress ◽  
Stalled Replication Forks

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

scholarly journals The evolutionary plasticity of chromosome metabolism allows adaptation to DNA replication stress

2019 ◽  
Author(s):  
Marco Fumasoni ◽  
Andrew W. Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Natural Populations ◽  
Replication Stress ◽  
Dna Damage Checkpoint ◽  
Whole Genome ◽  
Replication Forks ◽  
Evolutionary Trajectory ◽  
Chromatid Cohesion ◽  
Dna Replication Stress

AbstractChromosome metabolism is defined by the pathways that collectively maintain the genome, including chromosome replication, repair and segregation. Because aspects of these pathways are conserved, chromosome metabolism is considered resistant to evolutionary change. We used the budding yeast, Saccharomyces cerevisiae, to investigate the evolutionary plasticity of chromosome metabolism. We experimentally evolved cells constitutively experiencing DNA replication stress caused by the absence of Ctf4, a protein that coordinates the activities at replication forks. Parallel populations adapted to replication stress, over 1000 generations, by acquiring multiple, successive mutations. Whole-genome sequencing and testing candidate mutations revealed adaptive changes in three aspects of chromosome metabolism: DNA replication, DNA damage checkpoint and sister chromatid cohesion. Although no gene was mutated in every population, the same pathways were sequentially altered, defining a functionally reproducible evolutionary trajectory. We propose that this evolutionary plasticity of chromosome metabolism has important implications for genome evolution in natural populations and cancer.

Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks

DNA Repair ◽  
2012 ◽  
Vol 11 (9) ◽  
pp. 741-752 ◽  
Author(s):  
Eun-Mi Lee ◽  
Tram Thi Bich Trinh ◽  
Hee Jin Shim ◽  
Suk-Young Park ◽  
Trang Thi Thu Nguyen ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
G2 Arrest ◽  
Strand Breaks ◽  
Dna Replication Stress

scholarly journals Targeting FBXO44/SUV39H1 elicits tumor cell-specific DNA replication stress and viral mimicry

Cell Stress ◽  
2021 ◽  
Vol 5 (3) ◽  
pp. 37-39
Author(s):  
Jia Z. Shen ◽  
Charles Spruck
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
Replication Stress ◽  
Threshold Level ◽  
Cancer Therapies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Replication Stress ◽  
Viral Mimicry ◽  
New Generation

Repetitive elements (REs) are normally transcriptionally silenced in somatic cells by repressive epigenetic modifications, which are thought to include DNA methylation and histone modifications such as deacetylation, H3K9me3, and H4K20me3. Although, it is unclear how RE silencing is maintained through DNA replication cycles in rapidly growing cancer cells. On the other hand, the reactivation of endogenous retroelements beyond a threshold level of tolerance in cancer cells, such as by treatment with DNA demethylating agents or HDAC or LSD1 inhibitors, can induce viral mimicry responses that augment certain cancer therapies, including immunotherapy. However, these agents can also affect normal cells presenting obvious side effects. Therefore, uncovering cancer cell-specific RE silencing mechanisms could provide a basis for the development of a new generation of cancer immunotherapy drugs. In our study (Shen et al. (2020), Cell, doi: 10.1016/j.cell.2020.11.042), through a high-content RNAi screen we identified FBXO44 as a key regulator of H3K9me3-mediated transcriptional silencing of REs in cancer cells. Inhibition of FBXO44 or its co-factor SUV39H1 stimulated antiviral pathways and interferon (IFN) signaling and induced replication stress and DNA double-strand breaks (DSBs) in cancer cells, leading to restricted tumor growth and synergy with anti-PD-1 therapy (Figure 1).

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