Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468¿476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470¿476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation

Sangoma boy

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University Londo

Examples: What Teachers Are Doing With Poetry

In November, 112 teachers from across Indiana attended a full-day professional development workshop with renowned poet Georgia Heard. Here is a sampling of the things these teachers are now doing in their schools and classrooms as a result of that workshop

Public involvement in health research: a case study of one NHS project over 5 years

Background: Public involvement, both in the National Health Service (NHS) and in clinical research, is promoted as an important democratic principle. The declared aims are to reduce professional autocracy and allow a broader ownership of the research agenda; also to improve the design of, and recruitment and retention of patients to, clinical studies. There have been a number of national initiatives in the UK to improve public input to clinical research activities, but very few reports of effective and sustainable partnerships over time. This study reports the evaluation of one example, which is embedded in the NHS and university partnerships in the Norfolk area of England. Objectives Evaluate: • Putting principles into practice of public involvement in research over a 5 year period for one specific project (Patient and Public Involvement in Research). • How the model contributes to, and impacts upon, all stages of the research process. • Attitudes of the research community and lay volunteers to their mutual experiences of public involvement. • Key factors and strengths of this project, and areas for improvement. Methods: A mixed methods approach related to the 5 years from start of 2003 to end of 2007. This used descriptive statistics of volunteer activity, interviews with key stakeholders (13), questionnaires (53% response rate), and focus group with 10 volunteers to explore emergent themes. We analysed findings using a policy framework approach. Results: About 47 of the original 55 volunteers remained on the panel after 5 years. All have undertaken training, 38% have been involved in the full range of research activities offered, and 75% have attended at least one research project meeting. Some are active in governance, ethics, and advisory committees. Both the research community and the volunteers are very positive about the project. The researchers find it provides well prepared personnel, and gives a speedy and efficient way of fulfilling the expectations of funders for lay input. The volunteers find it gives them important opportunities to influence the quality of research and thus support improvements in patient care. Areas for improvement include increasing social diversity among the volunteers, and improving feedback on input from volunteers, without which volunteers tend to lose confidence and motivation. Conclusion: Long-term sustainable and valuable public input to research is possible. Key factors are committing resources, embedding the service in the infrastructure of a research consortium, and ongoing responsiveness by NHS staff and researchers. Additional activity to recruit and support access may be needed to attract people from a broad range of sociodemographic backgrounds. Some volunteers want more involvement than this model currently offers

Disrupting the Model: Fostering Cultural Change Through Academic Partnerships

Book chapter featured in the book, Affordable course materials: Electronic textbooks and open educational resources. This chapter highlights the University of Central Florida\u27s efforts and experiences related to textbook affordability and open educational resources

Chromosome breakage after G2 checkpoint release

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain

A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias

Fibrillin-1 is the major component of the 10–12 nm diameter extracellular matrix microfibrils. The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFβ dysregulation associated with these diseases