PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity

Supersensitive Multifluorophore RNA‐FISH for Early Virus Detection and Flow‐FISH by Using Click Chemistry

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid‐19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single‐fluorophore‐containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off‐target binding effects that create background noise. Here, we used click chemistry and alkyne‐modified DNA oligonucleotides to prepare multiple‐fluorophore‐containing probes. We found that these multiple‐dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection

DNA methylation and differential gene regulation in photoreceptor cell death

Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a twostage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-eta (pol eta), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of pol eta. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in similar to 30% of AML patients results in excessive degradation of pol eta. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a twostage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-eta (pol eta), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of pol eta. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in similar to 30% of AML patients results in excessive degradation of pol eta. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1

Организация электроснабжения электрохимической защиты магистральных трубопроводов

В процессе эксплуатации магистральные газопроводы (МГ), как и большинство других металлических подземных коммуникаций, подвергаются воздействию процессов коррозии. Интенсивность этих процессов, а, следовательно, и состояние МГ зависит от целого ряда факторов, к числу которых следует отнести: коррозионную активность и характеристики грунтов в местах прокладки газопровода, глубину залегания участков газопровода, пересечениями с ЛЭП высокого напряжения или с электрифицированной железной дорогой – источниками блуждающих токов, качеством изоляционного покрытия, наличием и эффективностью настройки средств электрохимической защитыIn the process of exploitation of magistral gas pipelines (MG), like most other metal pose of communication, are exposed to corrosion processes. The intensity of these processes, and, consequently, the condition of the person MG depends on a number of factors, among which include: corrosiveness and the characteristics of the grunts in the field strip, the gazebo, the depth of the selenium sites gazebo leading from Lap high voltage electrician or from the sale of honey for the presence of wandering well, the quality of the insulation coating, lithium and effective tuning of electrochemical protectio

Tumour hypoxia causes DNA hypermethylation by reducing TET activity

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylatio