PIKK-ing a New Partner: A New Role for PKB in the DNA Damage Response

The protein kinase PKB/Akt has long been associated with regulating signaling pathways that promote cell survival and cell growth, for example, in response to growth factors. In contrast, the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA damage and for cell survival after exposure to DNA-damaging agents, such as ionizing radiation. In a recent paper published in Molecular Cell, Hemmings and colleagues provide evidence that DNA-PK is required for the activation of PKB in response to exposure to ionizing radiation, suggesting that these two protein kinases may act together to promote survival after DNA damage

Health psychology’s role in sexual health care

This article provides an overview of the current and potential role of health psychology in sexual health care. Research, which applies various behaviour change techniques, is reviewed alongside one of the most extensively used theories, the Theory of Planned Behaviour (see Conner and Sparks 2009). This paper aims to link theory to practice giving the reader an indication of the range of work that health psychologists are involved in, and how this could be applied to sexual healthcare practice which supports positive behaviour change

Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody

We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor-DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region

Trapping Penguins with Entangled B Mesons

The first direct observation of time-reversal (T) violation in the $B\bar{B}$ system has been reported by the BaBar collaboration, employing the method of Ba$\tilde {\rm n}\!$uls and Bernab\'eu. Given this, we generalize their analysis of the time-dependent T-violating asymmetry ($A_{T}$) to consider different choices of CP tags for which the dominant amplitudes have the same weak phase. As one application, we find that it is possible to measure departures from the universality of $\sin(2\beta)$ directly. If $\sin(2\beta)$ is universal, as in the Standard Model, the method permits the direct determination of penguin effects in these channels. Our method, although no longer a strict test of T, can yield tests of the $\sin(2\beta)$ universality, or, alternatively, of penguin effects, of much improved precision even with existing data sets.Comment: 11 pages, 2 figures; refs. added; reframed with expanded discussions; version to appear in PLB; Metadata abstract updated onl

Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in vivo in activated T-cells

IL-2 gene expression in activated T-cells is initiated by chromatin remodeling at the IL-2 proximal promoter and conversion of a transcriptional repressor into a potent transcriptional activator. A purine-box regulator complex was purified from activated Jurkat T-cell nuclei based on sequence-specific DNA binding to the antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target DNA sequence in the proximal IL-2 promoter. ARRE DNA-binding subunits were identified as NF90, NF45 and systemic lupus erythematosis autoantigens, Ku80 and Ku70. Monoclonal antibodies to Ku80, Ku70 and NF90 specifically inhibit constitutive and inducible ARRE DNA-binding activity in Jurkat T-cells. Ku80, Ku70 and NF90 bind specifically to the IL-2 gene promoter in vivo, as demonstrated by chromatin immunoprecipitation. Activation of Jurkat T-cells and mouse primary spleen cells induces binding of Ku80 and NF90 to the IL-2 promoter in vivo, and decreases binding of Ku70 to the IL-2 promoter in vivo, and these dynamic changes are inhibited by immunosuppressants cyclosporin A and triptolide. Dynamic changes in binding of Ku80, Ku70 and NF90 to the IL-2 proximal promoter in vivo correlate with chromatin remodeling and transcriptional initiation in activated T-cells

Dissection of DNA double-strand-break repair using novel single-molecule forceps.

Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity

Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a “hinge” region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ