Abstract
Helicases are ubiquitious motor proteins essential in key biological processes which require single-stranded DNA (ssDNA) such as DNA replication, transcription, repair and recombination. In this thesis, various methods have been used to study the properties of the enzyme RepA and its function and mechanism.
RepA is the first intact hexameric replicative DNA helicase (from plasmid RSF1010) whose three-dimensional structure is known in detail. The six ATP binding sites of RepA are each located at the interfaces between two adjacent monomers and belong to Walker A and B-motifs, corresponding to motifs H1 and H2 of the DnaB-like helicase family. They are defined by the consensus sequence for the P loop 40GAGKS44 and residues Asp140, Glu77, His179 (belonging to the same monomer) and Arg207 (from the adjacent monomer).
Nucleotide binding to RepA shows a strong negative cooperativity. Although there are six potential ATP binding sites in the RepA hexamer, fluorescence measurements show that only three sites can be occupied by the ATP analogue TNP-ATP. RepA is more stable in the presence of ATPgS than in the presence of ADP (DDG = 0,29 kcal/mol), indicating that the additional phosphate group in ATPgS has a significant influence on RepA structure.
Circular dicroism, X-ray and electron microscopic studies show that nucleotide binding to RepA induces conformational changes. Fluorescence depolarization measurements further show dynamic segment movements around the ATP active site upon ATP binding, hydrolysis and ssDNA binding.
ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves both in the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA stimulated ATPase activity.
Mutation of Lys43 (Walker A motif) to alanine abolishes ATP binding and hydrolysis activity, indicating that this lysine is essential in the Walker A motif in DnaB-like helicase family.
Fluorescence correlation spectroscopy (FCS) and analytic ultracentrifugation were used to characterize the interaction of fluorescence labeled ssDNA with RepA. Equilibrium binding of ssDNA to RepA was only optimal in the presence ATPgS at pH 5.8. In the absence of ATPgS or at pH 7.6, complex formation between ssDNA and RepA was rather weak. Binding curves are compatible with one binding site for ssDNA present on RepA, with no indication of cooperativity.
Protein-DNA photo-crosslinking studies further show that only one subunit from the hexamer helicase is involved in the interaction with ssDNA at the same time and preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ssDNA.
RepA served as a model helicase to search for inhibitory compounds. The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibit the ATPase and dsDNA unwinding activities of RepA. Dimyricetin was the most effective inhibitor for both activities. Single-stranded DNA-dependent RepA ATPase activity is inhibited noncompetitively by all four compounds. Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species and these flavones may provide lead structures for the design of molecules helpful for unraveling the mechanism of helicase action and for the design of novel pharmacologically useful molecules.
A model is proposed in which RepA helicase sequencely hydrolyses ATP, translocates on ssDNA and unwinds DNA. |
