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Translesion DNA synthesis polymerase κ is essential to a carcinogen-induced nucleolar stress response

ABSTRACT: Goal of the experiment was to identify putative binding proteins enriched using the iPond technique.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Paolo Cifani

LAB HEAD: Paolo Cifani

PROVIDER: PXD038641 | Pride | 2024-07-25

REPOSITORIES: Pride

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			Action	DRS
	210923_SP_pool1.mzML	Mzml
	210923_SP_pool1.mzid.gz	Mzid
	210923_SP_pool1.xlsx	Xlsx
	210923_SP_pool1_1.raw	Raw
	210923_SP_pool1_2.raw	Raw

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Publications

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis.

Zhang Lifang L Braynen Janeen J Fahey Audrey A Chopra Kriti K Cifani Paolo P Tadesse Dimiru D Regulski Michael M Hu Fangle F van Dam Hubertus J J HJJ Xie Meng M Ware Doreen D Blaby-Haas Crysten E CE

Frontiers in plant science 20231025

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not ...[more]

PMID: 37965006

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Proteomic analysis of SYNCRIP deficient HSPCs

Project description:Alterations in SYNCRIP activity were found to alter gene transcription in leukemia cells. The effects of transcription alterations on expression of the corresponding proteins were investigated by LC/MSMS

2023-05-10 | PXD019779 | Pride

Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery.

Project description:DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.

| S-EPMC6251625 | biostudies-literature

Catalytic and non-catalytic functions of DNA polymerase kappa in translesion DNA synthesis

Project description:Translesion DNA synthesis (TLS) is a cellular process that enables the bypass of DNA lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (PolK) has the distinctive ability to bypass minor groove DNA adducts in vitro. However, PolK is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of PolK during lesion bypass. Strikingly, we show that PolK has two main functions during TLS, which are differentially regulated via Rev1 binding. On the one hand, PolK is essential to replicate across a minor groove DNA lesion in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, via its cooperative interaction with Rev1 and ubiquitylated PCNA, PolK appears to stabilize the Rev1-PolZ extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of PolK's catalytic activity. Together, our work identifies catalytic and non-catalytic functions of PolK in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases.

2024-08-27 | PXD044258 | Pride

Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase κ.

Project description:DNA polymerase (pol) κ efficiently catalyzes error-free translesion DNA synthesis (TLS) opposite bulky N2-guanyl lesions induced by carcinogens such as polycyclic aromatic hydrocarbons. We investigated the biochemical effects of nine human nonsynonymous germline POLK variations on the TLS properties of pol κ, utilizing recombinant pol κ (residues 1-526) enzymes and DNA templates containing an N2-CH2(9-anthracenyl)G (N2-AnthG), 8-oxo-7,8-dihydroguanine (8-oxoG), O6-methyl(Me)G, or an abasic site. In steady-state kinetic analyses, the R246X, R298H, T473A, and R512W variants displayed 7- to 18-fold decreases in kcat/Km for dCTP insertion opposite G and N2-AnthG, with 2- to 3-fold decreases in DNA binding affinity, compared to that of the wild-type, and further showed 5- to 190-fold decreases in kcat/Km for next-base extension from C paired with N2-AnthG. The A471V variant showed 2- to 4-fold decreases in kcat/Km for correct nucleotide insertion opposite and beyond G (or N2-AnthG) compared to that of the wild-type. These five hypoactive variants also showed similar patterns of attenuation of TLS activity opposite 8-oxoG, O6-MeG, and abasic lesions. By contrast, the T44M variant exhibited 7- to 11-fold decreases in kcat/Km for dCTP insertion opposite N2-AnthG and O6-MeG (as well as for dATP insertion opposite an abasic site) but not opposite both G and 8-oxoG, nor beyond N2-AnthG, compared to that of the wild-type. These results suggest that the R246X, R298H, T473A, R512W, and A471V variants cause a general catalytic impairment of pol κ opposite G and all four lesions, whereas the T44M variant induces opposite lesion-dependent catalytic impairment, i.e., only opposite O6-MeG, abasic, and bulky N2-G lesions but not opposite G and 8-oxoG, in pol κ, which might indicate that these hypoactive pol κ variants are genetic factors in modifying individual susceptibility to genotoxic carcinogens in certain subsets of populations.

| S-EPMC5116392 | biostudies-literature

Nucleolar RNA polymerase II drives ribosome biogenesis.

Project description:Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

| S-EPMC7486236 | biostudies-literature

Translesion Synthesis DNA Polymerase Kappa Is Indispensable for DNA Repair Synthesis in Cisplatin Exposed Dorsal Root Ganglion Neurons.

Project description:In the peripheral nervous system (PNS) in the absence of tight blood barrier, neurons are at increased risk of DNA damage, yet the question of how effectively PNS neurons manage DNA damage remains largely unanswered. Genotoxins in systemic circulation include chemotherapeutic drugs that reach peripheral neurons and damage their DNA. Because neurotoxicity of platinum-based class of chemotherapeutic drugs has been implicated in PNS neuropathies, we utilized an in vitro model of Dorsal Root Ganglia (DRGs) to investigate how peripheral neurons respond to cisplatin that forms intra- and interstrand crosslinks with their DNA. Our data revealed strong transcriptional upregulation of the translesion synthesis DNA polymerase kappa (Pol ?), while expression of other DNA polymerases remained unchanged. DNA Pol ? is involved in bypass synthesis of diverse DNA lesions and considered a vital player in cellular survival under injurious conditions. To assess the impact of Pol ? deficiency on cisplatin-exposed DRG neurons, Pol ? levels were reduced using siRNA. Pol ? targeting siRNA diminished the cisplatin-induced nuclear Pol ? immunoreactivity in DRG neurons and decreased the extent of cisplatin-induced DNA repair synthesis, as reflected in reduced incorporation of thymidine analog into nuclear DNA. Moreover, Pol ? depletion exacerbated global transcriptional suppression induced by cisplatin in DRG neurons. Collectively, these findings provide the first evidence for critical role of Pol ? in DNA damage response in the nervous system and call attention to implications of polymorphisms that modify Pol ? activity, on maintenance of genomic integrity and neuronal function in exogenously challenged PNS.

| S-EPMC5632588 | biostudies-literature

Mammalian DNA Polymerase Kappa Activity and Specificity.

Project description:DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

| S-EPMC6695781 | biostudies-literature

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

Project description:Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.

| S-EPMC9642814 | biostudies-literature

RNA polymerase II acts to promote the organization of chromatin following DNA replication

Project description:In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. The data uploaded are associated to the iPOND-TMT data uploaded under PXD046514 where protein re-association to newly replicated DNA was monitored upon transcription inhibition. To see whether changes observed in the iPOND-TMT time course experiments (PXD046514) are due to changes in overall protein abundance, whole extract samples were taken for each time point (Nascent, 1h and 2h after EdU labelling) and analysed by TMT-Mass Spec.

2024-02-14 | PXD040888 | Pride

RNA polymerase II acts to promote the organization of chromatin following DNA replication

Project description:In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. By performing iPOND-TMT time course experiments in the presents of transcription inhibition, we show that RNAPII promotes the re-association of hundreds of proteins with newly replicated DNA (Data uploaded under PXD046514). To show that this function of RNAPII is specific to newly replicated chromatin, an iPOND-TMT 24hr chase experiment was performed in the presence of transcription inhibition to examine its effect on the protein binding on steady state chromatin.

2024-02-14 | PXD046546 | Pride