Working with international collaborators the Dickman group have shown that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR–Cas systems. In contrast, the CRISPR–Cas type V-A system cleaves glucosyl-5-hydroxymethylated cytosine bases in DNA. These findings have important implications that could be exploited for genome engineering applications.
Vlot M, Houkes J, Lochs SJA, Swarts DC, Zheng P, Kunne T, Mohanraju P, Anders C, Jinek M, van der Oost J, Dickman MJ & Brouns SJJ (2017) Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes. Nucleic Acids Res.
The Dickman group have developed a method for the accurate quantification of nucleic acids using hypochromicity measurements in conjunction with UV spectrophotometry. Using this approach, we have measured the absorbance of both the unstructured and structured nucleic acids to accurately measure their hypochromicity and determine their extinction coefficients. For a range of different dsRNA, we have for the first time determined values of 46.18-47.29 μg/mL/A260 for the quantification of dsRNA using UV spectrophotometry. Moreover, this approach enables the accurate determination of the relative proportion of duplex nucleic acids in mixed ds/ss nucleic acid solutions, demonstrating significant advantages over current methods.
This work was performed in collaboration with Syngenta.
Nwokeoji AO, Kilby PM, Portwood DE & Dickman MJ (2017) Accurate Quantification of Nucleic Acids Using Hypochromicity Measurements in Conjunction with UV Spectrophotometry. Analytical Chemistry, 89(24), 13567-13574.
Genetic variants linked to an increased risk of breast cancer are described in two papers published this week in Nature and Nature Genetics. It is hoped that the genome-wide association studies, which collectively draw on data from hundreds of thousands of individuals, might lead to improved screening, earlier detection and better treatments for this disease.
Working with international collaborators, the Grasby lab have demonstrated how human flap endonuclease-1 verifies its correct DNA substrate by threading single-stranded DNA through a hole in protein. The threaded DNA flap is guided through the hole and positioned to react using a process the group have termed “phosphate steering”.
Phosphate steering by flap endonuclease 1 promotes 5´-flap specificity and incision to prevent genome instability. Susan E. Tsutakawa, Mark J. Thompson, Andrew S. Arvai, Alexander J. Neil, Steven J. Shaw, Sana I. Algasaier, Jane C. Kim, L. David Finger, Emma Jardine, Victoria J. B. Gotham, Altaf S. Sarker, Mai Z. Her, Fahad Rashid, Samir M. Hamdan, Sergei M. Mirkin, Jane A. Grasby and John A. Tainer (2017) Nature Communications 8, 15855 doi: 10.1038/ncom
The Grasby lab has shown that when exonuclease-1, a 5’-nuclease superfamily member, processes flapped DNAs it threads them through its helical arch. This threading process confines the enzyme reaction to discontinuous DNAs like those that have flaps. In contrast the reaction of continuous DNAs, such as the unbroken strand in gapped DNA is prevented on a biological timescale, thus preserving the genome.
Human Exonuclease 1 Threads 5′-Flap Substrates through Its Helical Arch. Steven J. Shaw, L. David Finger, and Jane A. Grasby (2017) Biochemistry, DOI: 10.1021/acs.biochem.7b00507
Congratulations to Mr Ola Rominiyi (clinical PhD student in the Collis laboratory) who has just been awarded a Royal College of Surgeon’s research Fellowship to support his studies centred on the development of novel DNA damage response targeting strategies in glioblastoma.
Long non-coding RNAs are crucial regulators of cellular processes in health and disease, but the functions of these often rare transcripts remain poorly understood. Using RNAseq data from the Utah Breast Cancer Study and The Cancer Genome Atlas, Angela Cox and James Bradford have identified a signature for basal breast cancer and delineated the impact of the surrounding healthy tissue on long non-coding RNA profiles.
Bradford JR, Cox A, Bernard P, Camp NJ. Consensus Analysis of Whole Transcriptome Profiles from Two Breast Cancer Patient Cohorts Reveals Long Non-Coding RNAs Associated with Intrinsic Subtype and the Tumour Microenvironment. PLoS One. 2016 Sep 29;11(9):e0163238. doi: 10.1371/journal.pone.0163238. PMID: 27685983.
In an article published in Science Advances, an international group of scientists led by Professor Sherif El-Khamisy has provided new insights into a novel repair pathway that controls how the mitochondrial DNA is read and processed, and how much oxygen the process requires. The results of the research have shown how the mitochondria is able to protect its DNA against damage caused by oxidative stress thereby keeping the cell healthy and functioning.
Dr. Collis, in collaboration with Prof. Thomas Helleday (Karolinska Institute, Sweden) and Dr. Patrick Eyers (University of Liverpool), has been awarded a Large Project grant from Weston Park Hospital Cancer Charity to support the development of novel small molecule inhibitors of CDK18 as potential future anti-cancer agents. This work builds on their functional characterisation of human CDK18, which was recently published in Nucleic Acids Research: doi.org/10.1093/nar/gkw615.
The Dickman group has developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.
This work was performed in collaboration with Syngenta.
Dr. Collis has been awarded a research grant from the Sheffield Hospitals Charity to help co-fund a clinical PhD project in his laboratory together with Brain Tumour Research and Support. This project is focused on finding new DNA damage response targeted drug combinations to improve the effectiveness of chemo- and radiotherapy regimes in drug resistant and incurable brain tumours.
Defenition Limited, a spin-out company from the University of Sheffield founded by Prof Jon Sayers, has agreed funding of up to £415,000 from IP Group plc, the developer of intellectual property-based businesses, to support the vital development of a new class of antibiotics. For full story see: www.sheffield.ac.uk/news/nr/sheffield-new-antibiotic-secures-funding-1.662057
Dr. Collis has been awarded a research grant from Brain Tumour Research and Support to help fund a clinical PhD project in his laboratory. This project is focused on finding new DNA damage response targeted drug combinations to improve the effectiveness of chemo- and radiotherapy regimes in resistant high-grade gliomas.
The Lambert group has recently reported a novel mechanism by which microRNA regulate the secretions of senescent fibroblasts. Fibroblasts, the major cellular component of the tumour microenvironment, become senescent with age and in response to the presence of tumour or treatments such as chemotherapy. These senescent cells are thought to increase the ability of cancer cells to metastasise by secreting proteins that support the spread of cancer cells. In their recent featured cover paper in Aging (dx.doi.org/10.18632/aging.100987), they report that a feedback loop including a microRNA and two proteins, PTEN and COX-2, regulate this secretion, identifying novel targets for drugs to disrupt the ability of fibroblasts to promote cancer spread.
The Collis group have just published a new paper which reveals that an ancient endogenised bornavirus gene that was integrated into our genome some 40 million years ago has developed key biological functions in human cells. Their work raises the possibility that other bornavirus-derived genetic elements might also have important biological functions. Their paper is freely available from the Nature publishing website at: www.nature.com/articles/srep35548.
The Grasby lab have published a paper in Nature Chemical Biology investigating the mode of action of inhibitors of human flap endonuclease 1. Working in collaboration with Astra Zeneca and Pelago Biosciences, it was revealed that FEN1 inhibitors blocked access to the active site, that binding of compounds to FEN1 could be detected in cells, and that cells where there is known synthetic lethality with FEN1 are much more sensitive to inhibition. For further information see www.sheffield.ac.uk/news/nr/blocking-enzymes-in-dna-damage-1.598305.
As part of a recruitment drive, Dr Tim Craggs has recently joined the University of Sheffield and SInFoNiA. Tim is a member of the Chemical Biology Cluster in the Department of Chemistry and works on using single molecule fluorescence to study nucleic acid processes.
The Dickman group has developed a rapid, versatile RNA extraction method free from phenol and chloroform. RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods and can be used to rapidly extract RNA from a variety of different cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.
This work was performed in collaboration with Syngenta.
The Collis group have just published a new paper in Cell Reports describing their identification and functional characterisation of a new component of the human DNA damage response. Their open access article can be downloaded freely at:
Professor Jon Sayers is lead author on a study published in Nature Structural and Molecular Biology that has revealed how branched DNA molecules are removed from the double-helical structure, a process crucial to how our DNA can replicate and repair itself.
Congratulations to SInFoNiA member Sherif El-Khamisy for his recent election as a Fellow of the Royal Society of Chemistry. This prodigious status is awarded to experienced researchers who have made an outstanding contribution to the advancement of chemical sciences.
The Collis group have just published a paper in Nucleic Acids Research describing their functional characterisation of human CDK18, and how it acts to facilitate efficient cellular responses to replication stress.
Breast, ovarian and prostate cancers are all hormone-related and have several features in common. The Breast, Ovarian and Prostate Cancer genetic consortia including Prof. Angela Cox’s group have recently carried out a large meta-analysis of these cancers in over 200,000 individuals. This study identified death receptor signalling and apoptosis as a key biological pathway in all three cancers, shedding new light on their shared biology.
Prof Mark Dickman (in collaboration with Prof Robert Read and Prof David Dockrell, Department of Infection, Immunity & Cardiovascular Disease) have recently published work developing a two dimensional-liquid chromatography (2D-LC) based approach for the identification and quantification of histone post translational modifications (PTMs) in conjunction with mass spectrometry (MS) analysis. 84 different histone peptide proteoforms with modifications at 18 different sites including combinatorial marks were identified, representing an increase of 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer. Their study makes a significant contribution to the analyse of changes in histone PTMs that are increasingly recognized as playing an important role in the pathogenesis of infectious diseases.
An invited review entitled “Ciliogenesis and the DNA damage response; a stressful relationship” by Institute co-director Dr. Spencer Collis has been published in the journal Cilia. The paper discusses new and emerging functional and genetic links between cellular responses to DNA damage and replication stress, and cilia-mediated stress signalling. This functional interplay has potential implications for the development and clinical management of human diseases such as ciliopathies, microcephalic disorders and cancer.
Congratulations to Dr Steve West who has been awarded and recently started a Wellcome Trust Investigator award entitled “Control, specificity and function of RNA polymerase II modification in human messenger RNA maturation”. The award valued at £911,621 will allow Dr West to use a multidisciplinary approach to investigate how transcription is coupled with mRNA maturation events in human cells. This is the second Wellcome Investigator award for SInFoNiA, the first being to Prof. Sherif El-Khamisy for his project entitled “The repair of oxidative and topoisomerase-induced chromosomal breaks: mechanisms and implications for human health”.
We are pleased to announce that Prof. Stuart Wilson has been awarded two BBSRC grants valued at £817,000 to study the process of mRNA transport in human cells. The first project will analyse how the cell regulates mRNA export in response to DNA damaging agents and the second will investigate how human cells decide which RNAs to retain in the nucleus and which to export to the cytoplasm.
Prof. Mark Dickman and colleagues have recently published work analysing how to separate nucleic acids. They investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. This work demonstrated that the pore size of the superficially porous silica particles significantly effects resolution. Furthermore, they have utilised 150 Å pore size solid-core particles to separate typical impurities generated in the synthesis of therapeutic oligonucleotides which are utilised as drugs to treat a wide range of diseases. This work was performed in conjunction with industrial partners GSK and Thermo Fisher Scientific.
Congratulations to Dr Bin Hu who has been awarded a Wellcome Trust Seed award valued at £99,868 to study “the molecular mechanism of chromosome condensation mediated by cohesin and condensin”.
Institute members Dr. Spencer Collis and Prof. Sherif El-Khamisy has been awarded a research grant from Brian Tumour Research and Support to help their collaborative PhD studentship project that aims to identify novel targets in chemo-resistant glioblastomas, for which there is currently no successful treatment.
Prof. Angie Cox reports that her group have recently proposed a new approach for genetic fine-mapping for common diseases like cancer, called discordant haplotype sequencing. This helps identify candidate functional variants by targetted highly parallel sequencing of small numbers of individuals who are homozygous for low-risk and high-risk genetic haplotypes. This is an alternative approach to the costly genotyping of thousands of individuals that is required, in traditional genetic fine-mapping of disease-associated variants.
We are pleased to announce that Institute co-director Dr. Spencer Collis has been promoted to Reader in Genome Stability within the Department of Oncology and Metabolism, to recognise his significant expertise and consistent contributions to this field.
Abhijit Patil (a PhD student from the Collis laboratory) has been awarded a HEFCE catalyst studentship to work in Cancer Research Technology’s discovery laboratories in Cambridge for a year. This will provide valuable experience and increases our ongoing collaborations with CRT. We wish Abhijit all the best in his placement,