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 Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pratibha Bhalla ◽  
Ashutosh Shukla ◽  
Dipti Vinayak Vernekar ◽  
Aneeshkumar Gopalakrishnan Arimbasseri ◽  
Kuljeet Singh Sandhu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Replication Fork ◽  
Replication Stress ◽  
Trna Genes ◽  
Replication Fork Progression ◽  
Pol I ◽  
Non Coding Rnas ◽  
Pol Iii

Abstract The RNA polymerase (pol) III transcribes mostly short, house-keeping genes, which produce stable, non-coding RNAs. The tRNAs genes, highly transcribed by pol III in vivo are known replication fork barriers. One of the transcription factors, the PAF1C (RNA polymerase II associated factor 1 complex) is reported to associate with pol I and pol II and influence their transcription. We found low level PAF1C occupancy on the yeast pol III-transcribed genes, which is not correlated with nucleosome positions, pol III occupancy and transcription. PAF1C interacts with the pol III transcription complex and causes pol III loss from the genes under replication stress. Genotoxin exposure causes pol III but not Paf1 loss from the genes. In comparison, Paf1 deletion leads to increased occupancy of pol III, γ-H2A and DNA pol2 in gene-specific manner. Paf1 restricts the accumulation of pol III by influencing the pol III pause on the genes, which reduces the pol III barrier to the replication fork progression.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

scholarly journals Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

2012 ◽  
Vol 33 (3) ◽  
pp. 571-581 ◽  
Author(s):  
Guoqi Liu ◽  
Xiaomi Chen ◽  
Michael Leffak
Keyword(s):  
Replication Fork ◽  
Trinucleotide Repeat ◽  
Cell Model ◽  
Replication Stress ◽  
Dna Hairpin ◽  
Replication Fork Stalling ◽  
Fork Stalling ◽  
Hairpin Formation ◽  
Lagging Strand

ABSTRACT(CTG)n· (CAG)ntrinucleotide repeat (TNR) expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)n· (CAG)nstructures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)45· (CAG)45causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)45· (CAG)45lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)45· (CAG)45expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linkedin vivo.

scholarly journals Mechanisms of Transcription-Replication Collisions in Bacteria

2005 ◽  
Vol 25 (3) ◽  
pp. 888-895 ◽  
Author(s):  
Ekaterina V. Mirkin ◽  
Sergei M. Mirkin
Keyword(s):  
Escherichia Coli ◽  
Direct Contact ◽  
Replication Origin ◽  
Replication Fork ◽  
Mutual Orientation ◽  
Physical Interaction ◽  
Inducible Promoters ◽  
Replication Fork Progression ◽  
Replication Intermediates

ABSTRACT While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.

scholarly journals KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance

Biology Open ◽  
2021 ◽  
Vol 10 (5) ◽  
Author(s):  
Solenne Gaillard ◽  
Virginie Charasson ◽  
Cyril Ribeyre ◽  
Kader Salifou ◽  
Marie-Jeanne Pillaire ◽  
...  
Keyword(s):  
Stress Response ◽  
Enzymatic Activity ◽  
Stress Tolerance ◽  
Ribonucleotide Reductase ◽  
Replication Stress ◽  
S Phase ◽  
Regulatory Subunit ◽  
Replication Forks ◽  
Histone Demethylases ◽  
Major Player

ABSTRACT KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of ribonucleotide reductase. Second, they are required for optimal levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

scholarly journals MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianning Lai ◽  
Ronan Broderick ◽  
Valérie Bergoglio ◽  
Jutta Zimmer ◽  
Sophie Badie ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
Replication Stress ◽  
Nuclease Activity ◽  
Dna Lesions ◽  
Genome Replication ◽  
Replication Fork Progression ◽  
Nucleolytic Activity ◽  
Genomic Regions

AbstractFailure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

scholarly journals The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

2019 ◽  
Author(s):  
Magdalena Ganz ◽  
Christopher Vogel ◽  
Christina Czada ◽  
Vera Jörke ◽  
Rebecca Kleiner ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Replication Fork ◽  
Replication Stress ◽  
Tumor Formation ◽  
Replication Forks ◽  
Functional Link ◽  
Protein Levels ◽  
Replication Fork Progression ◽  
Dna Replication Stress ◽  
Architectural Protein

ABSTRACTDNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

scholarly journals Loss of SIM2s inhibits RAD51 binding and leads to unresolved replication stress

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Scott J. Pearson ◽  
Jessica Elswood ◽  
Rola Barhoumi ◽  
Brittini Ming-Whitfield ◽  
Monique Rijnkels ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Replication Fork ◽  
Replication Stress ◽  
Genotoxic Stress ◽  
Mammary Epithelial ◽  
Breast Cancer Cell Lines ◽  
Genomic Integrity

Abstract Background Mutations in genes associated with homologous recombination (HR) increase an individual’s risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR. Methods Initial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s’ role in RAD51 recruitment was determined by immunoblotting. Results Here, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding. Conclusions Together, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.

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