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The mechanism of DNA cleavage by the ubiquitous H-N-H/bba-Me motif

Reference	BB/C515520/1
Principal Investigator / Supervisor	Professor Colin Kleanthous
Co-Investigators / Co-Supervisors
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	229,110
Status	Completed
Type	Research Grant
Start date	30/08/2005
End date	29/04/2009
Duration	44 months

Abstract

Horning endonucleases have played a key role in the evolution of genomes. Normally encoded within introns and inteins, they promote integration of mobile genetic elements into intronless or inteinless alleles by virtue of their specific cleavage of DNA in different genetic backgrounds, including mitrochrondiral, chloroplast, nuclear and bacteriophage genomes. Horning enzymes are typically classified into four groups based on active site consensus motifs: LAGLIDADG, GIY-Cys box and H-N-H. Over the last 5 years the metal-dependent mechanisms of all bar the H-N-H family have been structurally resolved. H-N-H DNases, originally described in group I and group II introns, have been identified across the three biological kingdoms, with enzymes containing this motif also found in a variety of other processes involving the endonucleolytic cleavage of DNA, including apoptosis, bacterial cell death and DNA mismatch repair. However, little is known as to how these enzymes of differing biological function but similar active site architecture cleave nucleic acid substrates. The present application builds on the most recent biochemical and structural data from my laboratory that suggest this ubiquitous motif to be an adaptable catalytic centre able to alkaline earth and transition metal ions to cleave nucleic acids by different mechanisms. Our work has focussed on DNase colicins, protein toxins from Escherichia coli whose specific role is to kill competing bacteria by degrading genomic DNA. The catalytic centre of a DNase colicin is an H-N-H motif which we have shown through previous work is structurally similar to a variety of other nucleases, all cast in different biological setting and revolving around endonucleolytic cleavage of DNA. The present proposal capitalises on past work showing that the mechanism and substrate specificity for this group of enzymes changes with different metal cofactors, as well as recently published crystallographic data on the colicin E9 DNase boundto dsDNA and metal ions, the first for this group of enzymes. The structures reveal unusual coordination chemistry to alkaline earth metal ions such as Mg2+. In this work we will (1) obtain further data on complexes of the enzyme bound to dsDNA (at higher resolution and using different mutants/forms of DNA): (2) determine structures of the enzyme bound to ssDNA, in the presence of transition metal ions which is the preferred cofactor with this substrate; (3) solve structure of the enzyme bound to RNA as the enzyme can also cleave RNA by a metal-independent mechanism; (4) determine microscopic pKa¿s for H-N-H motif residues in the presence of DNA and metal ions; (5) use steady-state and pre-steady state kinetics to define the kinetic mechanism of DNA hydrolysis by this ubiquitous group of enzymes.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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