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Biography
31/09/2024 to date Emeritus Professor, School of Chemistry
01/01/2008 to 30/09/2024 Professor of Biochemistry/Microbiology and Biological Chemistry.
04/11/2010 to 20/02/2011 Visiting Professor, MIT, Boston, USA.
01/09/2003 to 01/01/2008 Associate Professor and Reader in Biochemistry/Microbiology and Biological Chemistry
01/09/2000- 01/09/2003 Lecturer
01/10/1996 to 31/08/2000 Postdoctoral Research Associate (PDRA), The Wellcome Trust, Sir William Dunn School of Pathology, University of Oxford, UK
01/02/1995 to 30/09/1996 PDRA, The European Union, Institute of Molecular Biology and Biotechnology, University of Crete, Greece.
01/01/1994 to 31/12/1994 Obligatory Military Service, The Greek Army, Greece.
01/10/1990 to 31/12/1993 PDRA, The Wellcome Trust, Department of Biochemistry, University of Bristol, UK
1987 to 1990 PH.D. Funded by the Yorkshire Cancer Research Campaign (YCRC). Subject: Molecular Biology. Awarded: January 1991, The University of Sheffield, UK.
1984 to 1987 B.Sc. (Dual Honours) class ii(i), Biochemistry and Microbiology. Awarded: July 1987, The University of Sheffield, UK.
Expertise Summary
I was born and brought up in Greece and moved to the UK in 1982 as a 19 year old student to study biochemistry. With the exception of a short career break to do my military service (1994-96) in the Greek army, I have been living in the UK ever since. I worked throughout my career in the area of bacterial DNA replication, chromosome remodeling and DNA repair. I have the necessary knowledge, tools, authority, research expertise, track record, management experience and extensive network of national and international collaborators that collectively make me an ideal candidate for the Chair in Biochemistry in your department". I am applying a wide range of biochemical, biophysical, molecular biology, structural biology, single molecule and genetics techniques to probe structural/functional aspects of DNA replication enzymes, large macromolecular assemblies and chromosome remodeling, and have published 75 peer review papers/reviews.
Transcription and replication: I used AFM to show that autoregulation of the melR promoter involves four MelR molecules in a nucleoprotein complex that does not form DNA looping. Recently, I probed conflicts between replication and transcription in collaboration with Alan Grossman (MIT, Boston). Until my research was published in the journal Nature, the assumption in the field was that co-directional replication-transcription collisions are not detrimental to replication. My findings changed this view and revealed that such conflicts are serious enough to warrant the intervention of the replication restart machinery in a process that is PriA-dependent and DnaA-independent. This work was featured the spring 2011 edition of the BBSRC Business magazine.
Helicase translocation along the DNA: I used novel DNA backbone vinylphosphonate dinucleotide linkages to reveal the role of rotational backbone freedom on helicase translocation, as well as the action of nucleases. My group discovered that vinylphosphonate modifications restrict rotational backbone flexibility and inhibit DNA unwinding by DNA helicases (PcrA, the Bloom and Werner syndrome DNA helicases). I examined the effects of such linkages on the activities of exonuclease III, mung bean nuclease and DNA pol I, and discovered that tandem vinylphosphonate modifications inhibit the action of DNA pol I but do not confer resistance to nucleases. My group is now using such modifications to investigate RNA polymerase-DNA interactions.
The Bacillus subtilis helicase loader DnaI: My studies on the B. subtilis helicase loader DnaI established a functional interaction with the B. stearothermophilus DnaB helicase and discovered that this involves a Zn-coordinating module in the N-terminal domain of DnaI. In response to binding of the helicase the N-terminal domain acts as a molecular switch regulating access to the single stranded DNA binding site located in the C-terminal domain of DnaI.
The B. subtilis clamp-loader protein DnaX: My group provided the first AFM-images of the DnaB-DnaX complex and suggested a structural model. I revealed crucial residues for the B. subtilis DnaX pentamerization and helicase binding. I identified a Shine-Dalgarno sequence and a slippage site in the B.subtilis dnaX gene sequence that could induce transcriptional or translational slippage to produce a shorter polypeptide equivalent to the gram negative γ polypeptide. Furthermore, we have solved the crystal structure of the δ subunit of the B. subtilis clamp-loader complex and revealed by mass spectrometry that the complete hetero-pentameric τ3-δ-δ' clamp loader complex assembles via multiple pathways which differ from those exhibited by the E. coli clamp loader. Using mass spectrometry we also showed that the interaction of the τ3 subcomplex with the hexameric helicase is driven by ATP/Mg2+ conformational changes and hydrolysis of one ATP by the hexameric helicase is sufficient to stabilize the interaction with τ3.
The B. subtilis primosomal proteins DnaD and DnaB: I was the first to discover that the essential primosomal proteins of B. subtilis, DnaD and DnaB, possess global DNA remodeling activities. DnaD opens up supercoiled DNA and DnaB acts as a lateral compaction protein. DnaD remodels DNA by untwisting and stretching the double helix, eliminating writhe while keeping the linking number constant. Its remodeling function is the sum of a scaffold-forming activity residing in its N-terminal domain and a DNA-binding activity with a further DNA-dependent oligomerisation activity in the C-terminal domain. I solved the crystal structure of the scaffold forming N-terminal domain of DnaD and suggested a structural model for the scaffold. I determined the solution structure of the C-terminal domain and established the structural features that underpin its interaction with DNA. I identified a highly conserved YxxxIxxxW motif likely involved in DNA binding across all DnaD-like proteins. My bioinformatics studies uncovered a structural homology between the DnaD and DnaB replication initiation proteins suggesting that they have evolved from a common ancestral protein. I discovered that the DNA untwisting activity of DnaD stimulates Nth-mediated DNA repair targeting abasic sites.
The bacterial helicase (DnaB)-primase (DnaG) interaction: I solved the first solution structure of the C-terminal domain of the DnaG primase, responsible for its interaction with the DnaB helicase. The structure revealed a unique and surprising structural homology with the N-terminal domain of DnaB and suggested a mechanism that was probed further. I discovered that the activities of DnaB and DnaG in the complex are modulated by distinct but overlapping networks of residues, while domain swapping experiments revealed functional interchangeability between the C- and N-terminal domains of DnaG and DnaB, respectively. I isolated, by yeast three-hybrid screening of a random peptide library, an antagonist peptide that interferes with the DnaB-DnaG interaction. The recognition sites of the B. stearothermophilus and B. subtilis DnaG were found to be 5'-CTA-3', 5'-TTA-3' and 5'-TTT-3' and the starting ribonucleotide was established to be the ATP complementary to the middle thymine base of the recognition sites. These data have recently been confirmed by collaborative work with the Hinrich and Griep groups (Nebraska, USA), which also revealed that the C-terminal domain of DnaG is not only a structural module mediating the interaction with DnaB but is essential for optimal primer synthesis. I revealed the molecular basis of initiation specificity in bacteria primase enzymes. A further finding from my group showed that the activity of the bacterial primase is modulated allosterically by the clamp loader DnaX protein, and this modulation involves the18 C-terminal residues of DnaX.
The RepD-PcrA molecular motor: I was the first to develop an AFM-based assay to visualize the unwound products from helicase reactions using supercoiled plasmids. With this assay, I studied the directional loading and stimulation of the PcrA helicase by the plasmid-encoded replication initiator RepD. I discovered that PcrA is recruited onto the minus strand by a RepD that is covalently attached to the 5'-phosphate group at the nick site, within oriD. Subsequently, I monitored the entire unwinding of a supercoiled plasmid by fluorescence and found that unwinding is processive and directional, and occurs at a speed of ~60 bp/sec. I discovered that PcrA surprisingly is able to load at a nick independently of RepD but in this case it loads onto the plus strand and translocates non-processively in the opposite direction than that of the PcrA-RepD molecular motor. DNAaseI and ExoIII footprint studies were used to investigate the molecular details of the RepD-mediated PcrA loading at oriD and a novel mechanistic loading model was proposed.
Lagging strand replication in B. subtilis
I recapitulated the B. subtilis lagging strand replication system with purified recombinant proteins including the primase helicase loader DnaI, the helicase DnaC, the primase DnaG and the RNA-primer extending polymerase DnaE. I showed that these proteins interact to form a lagging strand specific replication sub-complex. The fidelity of DnaE is improved within this complex via allosteric interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaE. I identified the initiation specificity of the B. subtilis DnaG and showed that the primer hand-off from DnaG to DnaE takes place after de novo polymerization of only two ribonucleotides and does not require other replication proteins. Such hand-off requires direct interaction between the two proteins and I proposed a structural model for this complex based upon molecular modelling and docking studies guided yeast two hybrid interaction data.
Host pathogen interactions
I have also diversified my research efforts towards antimicrobial research and the understanding of host-pathogen interactions (see https://royalsociety.org/news/2013/bacteria-hijack/). I worked with academic and industrial collaborators towards developing the QPLEX /TYPLEX (Fe complexes) technology. I was instrumental in linking with industrial partners and collaborators to drive the commercialisation of this technology (see announcement by the FSA http://www.food.gov.uk/news-updates/campaigns/campylobacter/actnow/act-e-newsletter/banham-poultry-and-akeso-biomedical-trial-a-feed-additive-to-control-campylobacter-infection). This was a major focus of my research efforts studying the efficacy and molecular mechanism(s) of action of QPLEX/TYPLEX on different bacteria.
Human HELQ helicase
The human HELQ helicase is a superfamily 2, 3′-5 helicase homologous to POLQ and RNA helicases of the Ski2-like subfamily. It is involved in diverse aspects of DNA repair and is an emerging prognosis biomarker and novel drug target for cancer therapy. HELQ interacts with RPA through its inherently disordered N-HELQ domain and hence is recruited to RPA bound DNA substrates. We revealed a novel role for HELQ in R-loop resolution. We showed in cells and in vitro that HELQ is recruited by RPA at R-loops, which are then resolved if HELQ is catalytically active as an ATPase/helicase. Furthermore, we identifed a functional interaction of HELQ with XRN2, a nuclear 5′ to 3′ exoribonuclease, which we suggest coordinates R-loop unwinding by HELQ with RNA digestion by XRN2.
We assigned a new biological function for HELQ in genome stability in metazoans through its involvement with XRN2 in R-loop metabolism.