A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives

Nakazawa, Yuka; Yamashita, Shunichi; Lehmann, Alan R.; Ogi, Tomoo

doi:10.1016/j.dnarep.2010.01.015

Cited by 76 publications

(95 citation statements)

References 21 publications

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“…Recovery of RNA Synthesis (RRS) assay. Experimental details have been described previously 16 . Cells were plated in plastic 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were UV irradiated (10J/m 2 254nm UVC) and incubated for 12 h for RNA synthesis recovery. RRS levels were measured by the fluorescence-based ethynyluridine (EU)-incorporation assay 16 . Briefly, recovered cells were incubated for 2 h in media supplemented with 100µM 5-ethynyluridine (EU) followed by EU detection by copper-catalysed fluorescent-azide conjugation reaction (Click-reaction): Cells were fixed and permeabilised in 2% paraformaldehyde, 0.5% triton X-100 in PBS; after washing with PBS, cells were incubated with 15 µM Alexa Fluor 488 azide (Invitrogen) in 50mM…”

Section: Methodsmentioning

confidence: 99%

“…siRNA transfection was performed 72 h prior to the UDS assay. UDS levels were measured by the fluorescence-based ethynyldeoxyuridine (EdU)-incorporation assay 15,16 …”

Section: Methodsmentioning

confidence: 99%

“…Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig.…”

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Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

et al. 2012

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We initially performed exome-sequencing 11 of the two UV S S-A patients, Kps3 and XP24KO (details described in Methods, Supplementary Note, Table 2c). The patients were homozygous for c.367A>T mutation in UVSSA, which led to a premature termination, p.Lys123* (Fig. 1a, b). We identified the same homozygous mutation in Kps2 (sib. of Kps3), and a homozygous c.87delG, causing a frameshift p.Ile31Phefs*9, in an Israeli patient UV S S24TA (Fig. 1b, c, Supplementary Note, Supplementary Fig. 1). The identified mutations are summarized in Fig. 1d. We did not detect the 80kDa UVSSA protein in any of the UV S S-A patients (Fig. 1e). We additionally examined several mild xeroderma pigmentosum (XP) cases; in one such case, XP70TO 12 (Supplementary Table 1), we identified a homozygous p.Cys32Arg, in the UVSSA (Fig. 1c, d), implying that XP70TO is also in the UV S S-A group. The mutant protein was stably expressed in XP70TO cells (Fig. 1f, Supplementary Fig. 2a-d). 4Allele frequencies of the identified mutations in a control population were examined (Supplementary Note, Supplementary Fig. 3a). Haploinsufficiency for UVSSA is negligible as the parents of Kps2/Kps3 showed no symptoms 4 . In parallel with exome-sequencing, we performed whole-genome SNP-genotyping to identify runs-of-homozygosity (ROH) shared among the patients. We identified three overlapping-ROHs (> 1Mbps) on autosomes, one of which encompasses the UVSSA locus (Fig. 1g, Supplementary Table 3a, b, Supplementary Fig. 3b, c). No chromosome copy number variation was detected (Supplementary Fig. 3d).The above findings strongly suggest that the mutations in UVSSA in the UV S S-A patients are causal for the disease; we therefore, next examined the NER-activities in the UV S S-A cells (Fig. 2). Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig. 2c) caused a drastic reduction of RRS (Fig. 2d, Supplementary Fig. 4), whereas UDS was unaffected (Fig. 2e). Ectopic-expression of the wild-type UVSSA cDNA in UV S S-A cells restored normal RRS ( Fig. 2f; V5-tagged-UVSSA immunofluorescent-staining shown in Fig. 2g), while it did not affect RRS-level in normal, CS-A, or CS-B cells; neither ERCC8 nor ERCC6 cDNA expression in UV S S-A cells restored the RRS-level.We conclude that KIAA1530/UVSSA is the causal gene for UV S S-A.ERCC8 and ERCC6 genes are responsible for both CS and UV S S 7,8 . To evaluate whether UVSSA mutations may also result in CS-phenotypes, we sequenced 5 the UVSSA gene of 61 CS-patients whose genetic defects had not yet been determined (Supplementary Table 4). We found no obvious mutations except for four novel heterozygous changes. These changes as well as the SNPs, also found in control and UV S S-A individuals, do not affect the RRS-activity (Suppleme...

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“…Recovery of RNA Synthesis (RRS) assay. Experimental details have been described previously 16 . Cells were plated in plastic 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…siRNA transfection was performed 72 h prior to the UDS assay. UDS levels were measured by the fluorescence-based ethynyldeoxyuridine (EdU)-incorporation assay 15,16 …”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

et al. 2012

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“…At the cellular level, TC-NER or GG-NER deficiency results in UV hypersensitivity. As .90% of the UV-induced DNA lesions are processed via GG-NER, excision of lesions and the subsequent gap-filling DNA synthesis, measured outside S phase as unscheduled DNA synthesis or UDS, is only marginally affected in TC-NER-deficient cells as opposed to GG-NER-deficient cells (Limsirichaikul et al 2009;Nakazawa et al 2010). Direct monitoring of TC-NER is significantly more laborious; only strand-specific damage removal assays are able to directly measure this NER subpathway (Mellon et al 1987;Mellon 2005).…”

Section: The Subpathways Of Nermentioning

confidence: 99%

Mammalian Transcription-Coupled Excision Repair

Vermeulen¹,

Fousteri

2013

Cold Spring Harbor Perspectives in Biology

159

118

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Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process.

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Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells

Sarker

Trego

Zhang

et al. 2020

Environ and Mol Mutagen

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Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1 and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break (DSB) induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair (NER) exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

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A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives

Cited by 76 publications

References 21 publications

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

Mammalian Transcription-Coupled Excision Repair

Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells

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