Institute of Biological and Biomedical Sciences

Geoff Kneale

The Kneale Lab has extensive expertise in all aspects of biophysics, with the main focus on applications involving Protein-DNA interactions; our philosophy is that by combining structural and biophysical analysis with biochemical and functional studies, we will be able to build up a complete picture of the mechanism of action of the relevant macromolecules and their role in fundamental biological processes, particularly those related to gene expression and DNA replication.  Many of the biological systems we study are potential targets for antimicrobial drug design, and others are important diagnostic tools in the investigation of brain pathologies in prion diseases such as Alzheimer’s and in embryonic development.

For further details and publication history please see my staff profile

Our current laboratory research

Type I Restriction-Modification Enzymes

Type I Restriction-Modification Enzymes

Restriction-modification (R-M) systems recognise self from non-self DNA by modification (methylation) of their own DNA at specific sequences, protecting their DNA from cleavage by the cognate endonuclease. Type I enzymes are the most complex, being multi-subunit enzymes with both DNA methylation (MTase) and endonuclease (ENase) activity. They are molecular motors, hydrolysing ATP to translocate along DNA prior to cleavage. We have studied the shape and subunit organisation of the trimeric MTase (M2S) by small angle neutron scattering (SANS), utilizing selective deuteration and contrast variation to match out one of the subunits, and determined the first low-resolution structural model of these complex MTases. The larger ENase is encoded by three genes (R, M and S) and has 5 subunits (R2M2S). We have now determined  for the first time the molecular structures of a Type I RM enzyme, using electron microscopy (EM), small-angle scattering (neutron and X-ray), and molecular modeling (in collaboration with colleagues in Leeds, Edinburgh and Warsaw). We have shown that DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by EM, using an anti-restriction protein that acts as a DNA mimic.

The Structural basis of gene regulation

The Structural basis of gene regulation

We have long-standing interests in the molecular mechanisms that control gene expression, and how transcriptional regulator proteins  (both activators and repressors) recognize their cognate DNA binding sites with varying degrees of specificity.  A major interest is in the “controller” (C) proteins that regulate the expression of bacterial restriction- modification genes. Using DNA footprinting and gel retardation assays (EMSA), we identified the binding sites for the C protein at the promoters controlling the R-M genes. The C protein can act as either an activator or repressor, in a concentration dependent manner, thus controlling the timing of expression of these genes via an elegant genetic switch. In collaboration with Prof. K. Severinov at the Waksman Institute (Rutgers University, NJ, USA), we have demonstrated in vitro activation and repression of gene transcription by the C protein. Following biophysical analysis of the relevant DNA-protein complexes underpinning the switch, we have determined the 3D molecular structure of each of the complexes by X-ray crystallography, thereby elucidating the mechanism of R-M gene regulation and paving the way to understanding the principles of differential DNA sequence recognition.

Nucleosome Assembly Proteins

Nucleosome Assembly Proteins 

Histone chaperones direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange. They are therefore central to the regulation of multiple cellular processes, such as chromatin remodeling and DNA replication. The nucleosome assembly protein (NAP) family represents a key group of histone chaperones that are essential for cell viability. Early work in our lab defined the major domain of NAP and a combination of biochemical/biophysical analysis with studies on the function of the truncated NAP in Xenopus embryos led to an understanding of its role in development. Subsequently, we showed that NAP forms discrete multimers with histones H2A/H2B and H3/H4 at a stoichiometry of one NAP dimer to one histone dimer. These complexes have been characterized by size exclusion chromatography, analytical ultracentrifugation, multi-angle laser light scattering and small-angle x-ray scattering to reveal their oligomeric assembly states in solution. By employing single-particle cryo-electron microscopy, we visualized these complexes for the first time, revealing heterogeneous ring-like structures, potentially acting as large scaffolds for histone assembly and exchange.

Gene 5 Protein, tetraplex DNA and prions

Gene 5 Protein, tetraplex DNA and prions

Single-stranded DNA-binding proteins play a key role in DNA replication. The gene 5 protein (g5p) of filamentous bacteriophage fd is responsible for the switch from double-stranded to single-stranded viral DNA replication, following infection of the bacterial cell. Earlier work in our lab showed that the g5p preferentially binds to a specific 4-stranded (tetraplex) DNA structure that forms G-quartets and we have studied the relevant complexes by SAXS and Circular Dichroism. X-ray crystallographic studies of the protein-nucleic acid complexes are also in progress. In collaboration with Dr Wenquan Zou at the Department of Pathology, Case Western Reserve University School of Medicine (USA), we showed that g5p has an unusually high affinity for the infective form of the prion protein PrP. The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP(C) into its pathogenic isoform PrP(Sc 1). The conversion of PrP(C) to PrP(Sc) involves a conformational transition of alpha-helical to beta-sheet structure in the protein. However, the in vivo pathway is still poorly understood. The use of g5p to selectively enrich samples of infected brain tissue for PrP(Sc) is proving a unique tool in biomedical research for the analysis of prion-related diseases, such as CJD and Alzheimer’s.

Previous lab members

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Previous PhD Students and post-docs in the Kneale Lab PDF 190KB