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Nucleic Acids Research, 2002, Vol. 30, No. 18 4051-4060
© 2002 Oxford University Press

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus

P. Soultanas and D. B. Wigley*,1

School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK and 1 London Research Institute, Clare Hall Laboratories, Cancer Research UK, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK

*To whom correspondence should be addressed. Tel: +44 207 269 3930; Fax: +44 207 269 3803; Email: d.wigley{at}cancer.org.uk

Site-directed mutagenesis studies on conserved amino acid residues within motifs H1, H1a, H2 and H3 of the hexameric replicative helicase DnaB from Bacillus stearothermophilus revealed specific functions associated with these residues. In particular, residues that coordinate a bound Mg2+ in the active site (T217 and D320) are important for the function of the enzyme but are not required for the formation of stable hexamers. A conserved glutamic acid (E241) in motif H1a is likely to be involved in the activation of a water molecule for in line attack on the {gamma}-phosphate of the bound nucleotide during catalysis. A conserved glutamine (Q362) in motif H3 acts as a {gamma}-phosphate sensor and mediates the conformational coupling of nucleotide- and DNA-binding sites. The nature of the residue at this position is also important for the primase-mediated activation of DnaB, suggesting that primase uses the same conformational coupling pathway to induce its stimulatory effect on the activity of DnaB. Together, these mutations reveal a conservation of many aspects of biochemical activity in the active sites of monomeric and hexameric helicases.


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