Activity of DNA polymerase kappa across the genome in human fibroblasts
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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:DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp 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 to generate genome-wide repair maps. We have termed this method polymerase κ sequencing. Here, we present the human genome maps for Polκ activity in an undamaged cell line. We found that Polκ activity was enhanced in GC-rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.
| S-EPMC11252913 | biostudies-literature
Project description:DNA interstrand crosslinks (ICLs) are highly cytotoxic lesions that block essential cellular processes like replication and transcription. Endogenous ICLs can be induced by reactive aldehydes produced during normal cellular metabolism. Defective repair of these aldehyde-induced ICLs is associated with Fanconi anemia (FA), a cancer predisposition syndrome. We previously showed that acetaldehyde-induced ICLs are repaired by the Fanconi anemia (FA) pathway and a novel excision-independent pathway. Here, we demonstrate that ICLs induced by acrolein, another cellular aldehyde, are also repaired by both pathways, establishing the generality of aldehyde ICL repair. Focusing on the FA pathway, we identify DNA polymerase kappa (Polκ) as the primary translesion synthesis (TLS) polymerase responsible for the insertion step during lesion bypass of unhooked aldehyde ICLs. This function requires Polκ's catalytic activity and PCNA interaction domains, but is independent of Rev1 interaction. In contrast, Polκ has a minor, non-catalytic role in the extension step of cisplatin ICL repair that is dependent on Rev1 interaction. Our work reveals a key role for Polκ in aldehyde ICL repair and provides mechanistic insights into how different ICL structures determine the choice of TLS polymerases during repair.
2025-09-29 | PXD062134 | Pride
Project description:DNA interstrand crosslinks (ICLs) are highly cytotoxic lesions that block essential cellular processes like replication and transcription. Endogenous ICLs can be induced by reactive aldehydes produced during normal cellular metabolism. Defective repair of these aldehyde-induced ICLs is associated with Fanconi anemia (FA), a cancer predisposition syndrome. We previously showed that acetaldehyde-induced ICLs are repaired by the FA-pathway and a novel excision-independent pathway. Here, we demonstrate that ICLs induced by acrolein, another cellular aldehyde, are also repaired by both pathways, establishing the generality of aldehyde ICL repair. Focusing on the FA pathway, we identify DNA polymerase kappa (Polκ) as the primary translesion synthesis (TLS) polymerase responsible for the insertion step during lesion bypass of unhooked aldehyde ICLs. This function requires Polκ's catalytic activity and PCNA interaction domains but is independent of Rev1 interaction. In contrast, Polκ has a minor non-catalytic role in the extension step of cisplatin ICL repair that is dependent on Rev1 interaction. Our work reveals a key role for Polκ in aldehyde ICL repair and provides mechanistic insights into how different ICL structures determine the choice of TLS polymerases during repair.
2025-09-29 | PXD066315 | Pride
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.
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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:DNA polymerase iota (Pol ι) is a Y-family lesion bypass polymerases, characterized by its remarkably low fidelity. While Pol ι is ubiquitously expressed in nearly all cell types, its function remains largely undefined. Previous studies in cellular and mouse models deficient in Pol ι have revealed no discernible phenotype. In this report, we show that Pol ι plays an important role in mitotic DNA synthesis. Cells lacking Pol ι exhibit heightened vulnerability to replication stress, increased formation of micronuclei, and accumulation of chromosomal breaks. The protein level of Pol ι is tightly regulated and restricted to G2/M phase cells via CRL7/FBXW11-mediated proteasomal degradation. The absence of Pol ι during S phase ensures that its error-prone DNA synthesis activity is excluded from interfering with normal DNA replication. These findings suggest that Pol ι function is critical in resolving DNA damage-induced replication stress prior to the completion of mitosis.
2025-07-01 | PXD065672 |
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
2020-04-09 | GSE142708 | GEO
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