Activity of DNA polymerase kappa across the genome in human fibroblasts
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1081783 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28146873_1.fastq.gz | Fastqsanger.gz | |||
| SRR28146873_2.fastq.gz | Fastqsanger.gz | |||
| SRR28146874_1.fastq.gz | Fastqsanger.gz | |||
| SRR28146874_2.fastq.gz | Fastqsanger.gz | |||
| SRR28146875_1.fastq.gz | Fastqsanger.gz |
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Project description:DNA polymerase kappa (PolK) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replicating repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by PolK utilizing a PolK - specific substrate, N2-(4-ethynylbenzyl)-2′-deoxyguanosine (EBndG). After shearing of the DNA into 200-500bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne–azide cycloaddition reaction, and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method polymerase k sequencing (polK-seq). Here we present the human genome maps for pol k activity in an undamaged cell line. We found that pol k activity was enhanced in euchromatin regions, the promoter of genes, and in DNA that is replicated early in S-phase
2024-06-17 | GSE260451 | GEO
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
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
Project description:Using a chicken-hamster radiation hybrid panel (ChickRH6), we have mapped chicken chromosome(s) that contain possible factor(s) that permit avian polymerase activity, by increasing polymerase activity. We identified four hybrid clones permissive for polymerase activity. Activity was lost following 12 passages respectively in only one of the positive clones. Expression from the four positive radiation hybrid clones at passages 1, and 12 was measured and compared to expression of the hamster recipient line and 17 negative (for polymerase activity) clones.
2015-12-01 | E-MTAB-3643 | biostudies-arrayexpress
Project description:Type I interferon (IFN) is the first line of defense against virus infection. By using both in vivo and in vitro influenza infection models, we found that type I IFN-κ, limited the replication of influenza viruses by stimulating a IFNAR-MAPK-cFos-CHD6 axis. Similarly, Zika virus (ZIKV) was also highly sensitive to IFN-κ-mediated suppression. With an IAV infected mouse model, we found that IFN-κ was the earliest responding type I interferon among all known members in mice after H9N2 infection, a low-pathogenic Avian Influenza, whereas this early induction did not occur upon highly pathogenic H7N9 infection. IFN-κ can efficiently contain both low- and high-pathogenic influenza replication in cultured human lung cells, and CHD6 was the major effector responsive molecule for IFN-κ, but not for IFN-α/β. Furthermore, we discovered that both IFNAR1 and IFNAR2 subunits of type I interferon receptor and their downstream axis of p38-cFos are engaged in IFN-κ signaling cascade to acti vate CHD6, which didn`t require STAT1 activity. In addition, we showed that the pre-treatment with IFN-κ before IAV challenge protected mice from high mortality. Altogether, our study identified an IFN-κ-specific pathway that suppressed influenza A virus in vitro and in vivo. Thus, IFN-κ may have potential as a new prevention and treatment agents against emerging viruses
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Project description:Using doxycycline-inducible IFN-kappa expression in CIN612-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrate that IFN-kappa inhibits the growth of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited 4-6h after IFN-kappa expression. This was also observed with recombinant IFN-beta suggesting a common mechanism of IFNs. RNA-seq analysis identified 1367 IFN-kappa regulated genes of which 221 were modulated >2-fold. The majority of those (71%) matched known ISGs confirming that IFN-kappa acts as a bona fide type I IFN in hr-HPV-positive keratinocytes. RNAi and co-transfection experiments indicate that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-kappa. CIN612-9E/pInd-IFN-kappa were induced for 4h with 1µg/ml doxycyclin or not. Three biological replicates were analyzed.
2015-11-04 | E-GEOD-74658 | biostudies-arrayexpress
Project description:Using doxycycline-inducible IFN-kappa expression in CIN612-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrate that IFN-kappa inhibits the growth of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited 4-6h after IFN-kappa expression. This was also observed with recombinant IFN-beta suggesting a common mechanism of IFNs. RNA-seq analysis identified 1367 IFN-kappa regulated genes of which 221 were modulated >2-fold. The majority of those (71%) matched known ISGs confirming that IFN-kappa acts as a bona fide type I IFN in hr-HPV-positive keratinocytes. RNAi and co-transfection experiments indicate that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-kappa.
2015-11-04 | GSE74658 | GEO
Project description:Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. mRNAs were analyzed by two color microarray from two separate human neonatal dermal fibroblasts cell lines in proliferating, 7 days contact inhibition, or 14 days contact inhibition. Contact inhibited samples were co-hybridized to proliferating samples as a control, while an additional array co-hybridized the two proliferating samples to analyze reproducibility.
2012-11-30 | E-GEOD-42612 | biostudies-arrayexpress
Project description:Checkpoint-mediated DNA polymerase e exonuclease activity curbing counteracts resection-driven fork collapse
| PRJNA658081 | ENA
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