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

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

Evidence that prenatal care visit experiences influence perceptions of the child

Abstract Parents’ descriptions of their baby prenatally are associated with later caregiving behavior and attachment. We present two studies to investigate the role of prenatal care visits in shaping prenatal perceptions. In Study 1, 320 pregnant people provided a description of their baby, and at a follow-up (n = 173) reported on their toddler’s behavioral and emotional difficulties. Descriptors attributed to prenatal care visit experiences, versus other sources, had a more negative tone. More negative descriptions were prospectively associated with greater child difficulties. In Study 2, 161 people reported on the personality of a baby following an imagined prenatal care visit, in which participants were randomly assigned to conditions differing in statements made by the healthcare provider. Provider statements were associated with differences in perceptions of the fetus. Our findings provide evidence that prenatal care experiences influence perceptions of a child’s personality prior to birth, with potential consequences for later child functioning

Time perspective in adolescents and young adults: Enjoying the present and trusting in a better future

Time perspective is crucial in adolescence and youth, when individuals make important decisions related to their present and future. The focus of this research was to use the S-ZTPI scale in a sample of adolescents and young adults, and to analyse its associations with decision-making, relational styles and engagement. A structural equation model of the effects of S-ZTPI on these variables was computed, and its psychometric properties were found adequate. The results underline that young people’s present orientation is associated with a relational style based on confidence in oneself and others, and with active engagement in terms of responsibility and trust in a better future. Our findings suggest a positive description of adolescents’ views, as they are able to enjoy the time they are living in without giving up their responsibilities for making a better world for the future

Synthesis of DNA dumbbell based inhibitors for the human DNA methyltransferase Dnmt1.

(Figure Presented) Dumbbells that block: Dnmt1 is a crucial enzyme in maintaining the methylation pattern of genes and as such is a critical element of the epigenetic programming process. DNA dumbbell constructs have been developed that inhibit Dnmt1 and have potential in the regulation of DNA methylation patterns in cells (see scheme; SAM=S-adenosylmethionine, Fl=Cy3 fluorescence label, CN=5-azadC, C-Me=5-methyldC). © 2008 Wiley-VCH Verlag GmbH and Co. KGaA