Investigating the regulation of DNA non-homologous end-joining through Ku70/80 interacting factors

DNA double-strand breaks are the most deleterious type of DNA damage that cells experience, which makes the study of double-strand break repair extremely important. Unrepaired or aberrantly repaired DNA can result in changes to core genes with critical function and thus lead to multiple diseases. Two main repair pathways for double-strand breaks exist: homologous recombination (HR) and non-homologous end-joining (NHEJ). Whilst the regulation of HR has been heavily investigated, the regulation of NHEJ remains to be fully explored. The aim of this thesis is to investigate the regulation of DNA NHEJ through interacting factors of the core NHEJ protein heterodimer, Ku70/80 (Ku). This thesis consists of three main research projects. The first, explores the potential role of the CUL4 substrate adaptor, WDR76, in the removal of Ku from sites of DNA damage. Data presented here highlight a role of WDR76 in the DNA damage response (DDR), and through effects on Ku removal kinetics, suggest a role for WDR76 in the regulating NHEJ. The second research project investigates a potential cyclin-dependent kinase phosphorylation site on the protein paralog of XRCC4 and XLF (PAXX). As PAXX is a Ku interactor with a role in NHEJ, the effect of PAXX phosphorylation is investigated as a potential NHEJ regulatory system. Lastly, I investigate the role of the RecQ helicase WRN, whose precise roles in the DDR are unclear. As an interactor of both HR and NHEJ proteins, WRN may affect the regulation of both pathways. WRN knockout cells were generated and a CRISPR-Cas9 screen performed to identify suppressors of WRN sensitivity to DNA damage. The targets identified offer insights into WRN function.BBSRC Horizon Discovery Steve Jackson Laborator

Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination.

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.Cancer Research UK (Grant IDs: C6/A18796, C6946/A14492, C6/A18796), European Research Council (Grant ID: 310917), Wellcome Trust (Grant ID: WT092096), University of Cambridge, Institut PasteurThis is the final version of the article. It first appeared from Elsevier (Cell Press) via http://dx.doi.org/10.1016/j.celrep.2016.08.06

Horner–Wadsworth–Emmons olefination of proteins and glycoproteins

Acknowledgements: We acknowledge funding by the Engineering and Physical Sciences Research Council (EPSRC), Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca plc under the Prosperity Partnership grant no. EP/S005226/1. We are grateful to M. Cliff, R. Spiess, M. Papworth and T. Murray for their support with 19F-NMR, MS analyses and antibody glycoengineering discussions.Funder: EPSRCAbstractChemo-selective modifications of proteins are fundamental to the advancement of biological and pharmaceutical sciences. The search for biocompatible chemical reactions has prompted us to investigate Horner–Wadsworth–Emmons (HWE) olefinations, iconic reactions in organic synthesis that would give rise to new selective protein olefinations. Our choice of HWE olefinations was inspired by the growing number of methods for generating aldehydes as transient reactive groups in proteins and the potential for mild and simple reaction conditions. Here we show that HWE olefination reactions on aldehydes, produced by both chemical and enzymatic methods, are compatible with physiological conditions and highly selective in small and large proteins, including therapeutic antibodies and stable recombinant proteins exemplified by green fluorescent protein. Reaction kinetics can be fine-tuned over orders of magnitude both by judicious use of substituents and pH regulation. The electrophilic nature of the HWE olefination products can be tuned to allow for subsequent nucleophilic additions, including thiol- and phospha-Michael additions. Our results demonstrate that HWE olefination of aldehydes in proteins provides efficient and selective bioconjugation chemistries that are orthogonal to existing methods.</jats:p

Horner–Wadsworth–Emmons olefination of proteins and glycoproteins

Acknowledgements: We acknowledge funding by the Engineering and Physical Sciences Research Council (EPSRC), Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca plc under the Prosperity Partnership grant no. EP/S005226/1. We are grateful to M. Cliff, R. Spiess, M. Papworth and T. Murray for their support with 19F-NMR, MS analyses and antibody glycoengineering discussions.Funder: EPSRCChemo-selective modifications of proteins are fundamental to the advancement of biological and pharmaceutical sciences. The search for biocompatible chemical reactions has prompted us to investigate Horner–Wadsworth–Emmons (HWE) olefinations, iconic reactions in organic synthesis that would give rise to new selective protein olefinations. Our choice of HWE olefinations was inspired by the growing number of methods for generating aldehydes as transient reactive groups in proteins and the potential for mild and simple reaction conditions. Here we show that HWE olefination reactions on aldehydes, produced by both chemical and enzymatic methods, are compatible with physiological conditions and highly selective in small and large proteins, including therapeutic antibodies and stable recombinant proteins exemplified by green fluorescent protein. Reaction kinetics can be fine-tuned over orders of magnitude both by judicious use of substituents and pH regulation. The electrophilic nature of the HWE olefination products can be tuned to allow for subsequent nucleophilic additions, including thiol- and phospha-Michael additions. Our results demonstrate that HWE olefination of aldehydes in proteins provides efficient and selective bioconjugation chemistries that are orthogonal to existing methods

Horner–Wadsworth–Emmons olefination of proteins and glycoproteins

Chemo-selective modifications of proteins are fundamental to the advancement of biological and pharmaceutical sciences. The search for biocompatible chemical reactions has prompted us to investigate Horner–Wadsworth–Emmons (HWE) olefinations, iconic reactions in organic synthesis that would give rise to new selective protein olefinations. Our choice of HWE olefinations was inspired by the growing number of methods for generating aldehydes as transient reactive groups in proteins and the potential for mild and simple reaction conditions. Here we show that HWE olefination reactions on aldehydes, produced by both chemical and enzymatic methods, are compatible with physiological conditions and highly selective in small and large proteins, including therapeutic antibodies and stable recombinant proteins exemplified by green fluorescent protein. Reaction kinetics can be fine-tuned over orders of magnitude both by judicious use of substituents and pH regulation. The electrophilic nature of the HWE olefination products can be tuned to allow for subsequent nucleophilic additions, including thiol- and phospha-Michael additions. Our results demonstrate that HWE olefination of aldehydes in proteins provides efficient and selective bioconjugation chemistries that are orthogonal to existing methods. (Figure presented.)</p