The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding abilit

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity

Novel immunohistochemistry-based signatures to predict metastatic site of triple-negative breast cancers

Background: Although distant metastasis (DM) in breast cancer (BC) is the most lethal form of recurrence and the most commonunderlying cause of cancer related deaths, the outcome following the development of DM is related to the site of metastasis.Triple negative BC (TNBC) is an aggressive form of BC characterised by early recurrences and high mortality. Athough multiplevariables can be used to predict the risk of metastasis, few markers can predict the specific site of metastasis. This study aimed atidentifying a biomarker signature to predict particular sites of DM in TNBC.Methods: A clinically annotated series of 322 TNBC were immunohistochemically stained with 133 biomarkers relevant to BC, todevelop multibiomarker models for predicting metastasis to the bone, liver, lung and brain. Patients who experienced metastasisto each site were compared with those who did not, by gradually filtering the biomarker set via a two-tailed t-test and Coxunivariate analyses. Biomarker combinations were finally ranked based on statistical significance, and evaluated in multivariableanalyses.Results: Our final models were able to stratify TNBC patients into high risk groups that showed over 5, 6, 7 and 8 times higher riskof developing metastasis to the bone, liver, lung and brain, respectively, than low-risk subgroups. These models for predictingsite-specific metastasis retained significance following adjustment for tumour size, patient age and chemotherapy status.Conclusions: Our novel IHC-based biomarkers signatures, when assessed in primary TNBC tumours, enable prediction of specificsites of metastasis, and potentially unravel biomarkers previously unknown in site tropism

Hypomethylation of the c-myc promoter region induced by phenobarbital in rat liver

Background. The changes in DNA methylation are considered as one of the early events in hepatocarcinogenesis. Objective. We evaluated the ability of phenobarbital (PB) – the most widely used anticonvulsant worldwide and classical rodent liver carcinogen – to cause the promoter region of the c-myc protooncogene hypomethylation as well as changes of mRNA level of this gene. Moreover, the expression of Dnmt1 protein in rat treated with this compound was analyzed. Material and Methods. Male Wistar rats received PB in daily oral doses of 92.8 mg kg-1 b.w. day-1 (at 24-h intervals; for one, three and fourteen days). Methylation of the c-myc promoter region was measured by PCR-based methylationsensitive restriction enzyme analysis (MSRA). Levels of mRNA for c-myc and protein Dnmt1 were assayed using Real- Time PCR and Western Blot, respectively. Results. The study showed that phenobarbital stimulated persistent changes in DNA methylation, i.e. loss of methylation in the promoter region of the c-myc gene and up-regulated its mRNA level. In addition, a significant increase in protein level of Dnmt1 in the c-myc over-expressing liver cells was observed. Conclusion. The oppose relationship between Dnmt1 activity and methylation status of c-myc gene was demonstrated. The c-myc over-expression by demethylation might represent an important, early events in the mechanism of action (MOA) of phenobarbital.Wprowadzenie. Zmiany metylacji DNA są rozważane jako jeden z wczesnych mechanizmów hepatokancerogenezy. Cel pracy. Celem badań była ocena wpływu fenobarbiatlu (PB) - leku przeciwpadaczkowego, modelowego promotora raka wątroby - na poziom metylacji regionu promotorowego i ekspresji na poziomie mRNA protoonkogenu c-myc. Ponadto dokonano analizy poziomu ekspresji białka Dnmt1. Materiał i metody. Samce szczurów szczepu Wistar otrzymywały PB w dawce 92,8 mg/kg m.c. x dzień-1 jednorazowo, 3-krotnie i 14-krotnie. Ocenę poziomu zmian metylacji genu c-myc dokonano metodą MSRA (ang. Methylation-Sensitive Restriction Enzyme Analysis). Analizę względnego poziomu transkryptów genu c-myc i białka Dnmt1 przeprowadzano odpowiednio metodą PCR w czasie rzeczywistymi i techniką Western Blot. Wyniki. W wyniku oddziaływania fenobarbitalu wykazano trwałe zmiany metylacji DNA - obniżenie metylacji w rejonie promotorowym genu c-myc i nadekspresję badanego genu na poziomie mRNA. Jednocześnie obserwowano statystycznie istotny wzrost poziomu białka Dnmt1. Wnioski. Wykazano odwrotną zależność między aktywnością Dnmt1 a poziomem metylacji genu c-myc. Nadekspresja c-myc w wyniku demetylacji może stanowić istotne, wczesne zdarzenie w mechanizmie działania (ang. Mechanism of Action (MOA)) fenobarbitalu

Supramolecular cylinders target bulge structures in the 5′ UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5′ UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents

Aberrant repair of etheno-DNA adducts in leukocytes and colon tissue of colon cancer patients

International audienceTo assess the role of lipid peroxidation-induced DNA damage and repair in colon carcinogenesis, the excision rates and levels of 1,N-6-etheno-2'-deoxyadenosine (epsilon dA), 3,N-4-etheno-2'-deoxycytidine (epsilon dC), and 1,N-2-etheno-2'-deoxyguanosine (1,N-2-epsilon dG) were analyzed in polymorphic blood leukocytes (PBL) and resected colon tissues of 54 colorectal carcinoma (CRC) patients and PBL of 56 healthy individuals. In PBL the excision rates of 1,N-6-ethenoadenine (epsilon Ade) and 3,N-4-ethenocytosine (epsilon Cyt), measured by the nicking of oligodeoxynucleotide duplexes with single lesions, and unexpectedly also the levels of epsilon dA and 1,N-2-epsilon dG, measured by LC/MS/MS, were lower in CRC patients than in controls. In contrast the mRNA levels of repair enzymes, alkylpurine- and thymine-DNA glycosylases and abasic site endonuclease (APE1), were higher in PBL of CRC patients than in those of controls, as measured by QPCR. In the target colon tissues epsilon Ade and epsilon Cyt excision rates were higher, whereas the epsilon dA and epsilon dC levels in DNA, measured by P-32-postlabeling, were lower in tumor than in adjacent colon tissue, although a higher mRNA level was observed only for APE1. This suggests that during the onset of carcinogenesis, etheno adduct repair in the colon seems to be under a complex transcriptional and posttranscriptional control, whereby deregulation may act as a driving force for malignancy

Supramolecular Cylinders Target Bulge Structures in the 5′ UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication**

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5′ UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents