RESEARCH INTERESTS:

The studies described below are conducted in collaboration with research groups in Canada, Poland and USA, and were funded by the BBSRC, Nuffield Foundation, NATO and the Royal Society.

Our research has focused on three areas related to biochemical analysis of DNA metabolism.

1)  Genetic stability of repeating DNA sequences. Unusual mutation events involving  the expansion of specific DNA triplet repeat sequences have been identified as the cause of at  least 14 human hereditary disorders, including Huntington's, myotonic dystrophy and various types  of ataxia. Biochemical events that generate these mutations are only partially understood, but  are probably due to interactions between unusual DNA structures and reactions occurring on DNA  [Bowater & Wells (2001) Prog. Nucl. Acids Res. & Mol. Biol., 66, 159-202  Pubmed Abstract]. We have established a model system that allows analysis of molecular mechanisms  leading to genetic instability of DNA repeats in bacteria. We have initiated studies to determine  how triplet repeat stability is influenced by various aspects of DNA metabolism, including  transcription, DNA repair and DNA structure. Recently, we have shown that the mismatch repair  pathway in bacteria promotes large deletions in long DNA triplet repeats [Parniewski et al.  (2000) J. Mol. Biol., 299, 865-874 PubMed Abstract].  These studies are exciting because they demonstrate that  the mismatch repair pathway can promote genomic instability, in contrast to it's usual role  within the cell. Our studies were funded by HFSP, NATO, the Royal Society and the Wellcome Trust and are  conducted in collaboration with various research groups, including:
Dr. Pawel Parniewski, Center of Microbiology & Virology, Polish Academy of Sciences, Lodz, Poland, e-mail: parniewski@cmiwpan.lodz.pl
Dr. Robert Wells, Center for Genome Research, Institute of Biosciences and Technology, Texas Medical Center, 2121 W. Holcombe Blvd., Houston, USA, e-mail:rwells@ibt.tamu.edu
Dr. Christopher Pearson, The Hospital for Sick Children, Department of Genetics, 555 University Avenue, Toronto, Ontario, Canada, e-mail: cepearson@genet.sickkids.on.ca



2)  Macromolecular interactions of BRCT domains. First discovered in the breast  cancer susceptibility gene, BRCA1, BRCT domains have been identified in many DNA repair  proteins from different organisms. It seems likely that these domains are utilised during  macromolecular interactions (protein-protein and/or protein-DNA). To characterise the role  of BRCT domains in such complexes, we are performing experiments in two model organisms,  Arabidopsis thaliana and Escherichia coli. The studies of A. thaliana are being performed  in collaboration with Dr. Mark Coleman (School of Biological Sciences, UEA).  Within the genome of A. thaliana , we have identified 14 open reading frames that contain  BRCT domains. For details of all predicted DNA repair proteins in A. thaliana  click here. Biochemical and molecular  biological studies have been initiated to characterise macromolecular interactions co-ordinated  by some of the BRCT domains identified in A. thalaina. Our second approach to investigate BRCT  domains is to focus on the specialised BRCT domain of E. coli DNA ligase. The central  aim of our study is to perform in vivo and in vitro analysis to determine  the involvement of the BRCT domain in essential protein-protein and DNA-protein interactions  of the DNA ligase. Structural analysis of E. coli DNA ligase is being performed in  collaboration with Dr. Andrew Hemmings (School of Biological Sciences & School of Chemical  and Pharmacy, UEA).In collaboration with   Drs. Julea Butt   and Andrew Mayes (School of Biological Sciences & School of Chemical and Pharmacy, UEA) we are  also developing novel techniques to study DNA-protein interactions. 



3)  Eubacterial DNA ligases. Recently, examination of microbial genome sequences has  suggested that some eubacteria may contain two different types of DNA ligases [Wilkinson,  Day & Bowater (2001) Molec. Microbiol., 40, 1241-1248]. Analogous to the situation in  eucaryotes, this observation suggests that the different DNA ligases may be required for  various aspects of DNA metabolism, such as replication, repair and recombination.. To  understand the requirement for multiple eubacterial DNA ligases, we have initiated studies of  the multiple potential DNA ligases identified in the genomes of Streptomyces coelicolor and  Mycobacterium tuberculosis and in the soon-to-be completed genome of Rhizobium leguminosarum.  Our collaborators in these studies are:
DNA ligases of S. coelicolor: Dr. Gabriella Kelemen (School of Biological Sciences, UEA) and Dr. Tobias Kieser (John Innes Centre, Norwich, UK).
DNA ligases of M. tuberculosis:   Dr. Aidan Doherty (Genome Damage & Stability Centre, University of Sussex, Brighton, UK).
DNA ligases of R. leguminosarum: Prof. Andy Johnston (School of Biological Sciences, UEA).

Click here to open a table of all DNA ligases identified in eubacterial genomes



4)  RNA ligases. Biochemical understanding of RNA ligases is less developed than that of DNA ligases, but the mechanism of phosphodiester bond formation is believed to be similar in both types of enzymes. RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (Rnl1, gp63) is the best-studied member of this class of enzymes. Recently, T4 Rnl2 has been identified, which exemplifies a distinct ligase family, defined by variant motifs [Ho & Shuman, 2002, PNAS, 99, 12709-14]. Using molecular biological and biochemical techniques, we are aiming to identify what dictates the different substrate specificities of different nucleic acid ligases. Detailed understanding of RNA ligases will improve their potential as molecular biological tools to be used in studies of the role of RNA sequence and structure in gene expression e.g. through RNA inhibition (RNAi) and ribozymes.