Nonhomologous end joining: A good solution for bad ends

Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy

Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons

Essential role for polymerase specialization in cellular nonhomologous end joining

Nonhomologous end joining (NHEJ) is a DNA double-strand break repair pathway required for development of the adaptive immune response, maintenance of cellular proliferative capacity, and the response to several commonly used cancer treatments. A major challenge faced by this pathway is that chromosome breaks can have dirty end structures, making them difficult to repair. We show here that two mammalian DNA polymerases have an unexpectedly pivotal role in helping resolve such ends. Each is proficient in different contexts and has a differing impact on repair fidelity. This work sheds light on how NHEJ has evolved to be flexible during repair and identifies two polymerases as critical for this process

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures, but it is unclear how the steps employed for resolution are determined. We sought to address this question by analyzing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85% to 6%. End-processing by nucleases and polymerases is increased to compensate, though paths with the fewest number of steps to generate a substrate suitable for ligation are favored. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors

Essential role for polymerase specialization in cellular nonhomologous end joining

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) μ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol μ using the unpaired base adjacent to the downstream 5′ phosphate even when there are available template sites further upstream of this position; this makes Pol μ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol μ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.This research was supported by National Cancer Institute [Grant CA097096 (to D.A.R.)]; Postdoctoral Fellowship PF-14-0438-01-DMC from the American Cancer Society (to J.M.P.); Ministerio de Ciencia y Tecnologia [Grant BFU2012-37969 (to L.B.)] and a European Molecular Biology Organization fellowship (to A.A.).Peer Reviewe

Becoming a Trainer: The Experience of Philippine English Teachers in the Primary Innovations Project

In an era when English has been spreading exponentially (Crystal, 1997; Graddol, 1997, 2006), a development linked to economic expansion and greater access to knowledge, the role of competent teacher trainers is crucial in facilitating teacher development programs (Burns and Richards, 2009) that can enhance expertise among English teachers so that they can respond more fully to the pedagogic demands of global English. However, relatively little is known about the process of becoming a teacher trainer. This chapter reflects on the experience of four skillful English teachers in the Philippines as they learned how to become teacher trainers in the Primary Innovations Project (PIP). First, I shall give an account of PIP against the background of English teacher education in the Philippines, including a profile of the participants of the study — the Primary Trainers of Teachers (PToTs), and an analysis of PIP content and procedures. Then I describe the PToTs’ experiences of becoming trainers, particularly how they accessed their teaching and training skills and what they learned from such experiences, based on a small-scale inquiry I undertook. I conclude by extracting learning points from the reflection on PToTs’ experiences in relation to the dynamics of ELT trainer development processes and by discussing the implications for the development of thinking on professional learning and the training of teacher trainers

Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons

Linguistic Issues in Facial Animation

Our goal is to build a system of 3D animation of facial expressions of emotion correlated with the intonation of the voice. Up till now, the existing systems did not take into account the link between these two features. We will look at the rules that control these relations (intonation/emotions and facial expressions/emotions) as well as the coordination of these various modes of expressions. Given an utterance, we consider how the messages (what is new/old information in the given context) transmitted through the choice of accents and their placement, are conveyed through the face. The facial model integrates the action of each muscle or group of muscles as well as the propagation of the muscles' movement. Our first step will be to enumerate and to differentiate facial movements linked to emotions as opposed to those linked to conversation. Then, we will examine what the rules are that drive them and how their different functions interact. Key words: facial animation, emotion, intona..