Published online 15 June 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 3190-3197
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved
Structure-specific DNA binding and bipolar helicase activities of PcrA
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, USA
* To whom correspondence should be addressed. Tel: +1 412 648 9025; Fax: +1 412 624 1401; Email: Khan{at}pitt.edu
Received April 26, 2004; Revised and Accepted May 15, 2004
PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5'
3' and 3'5' helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5' or 3' single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity to partially duplex DNA containing hairpin structures adjacent to a 6 nt long 5' single-stranded region and one unpaired nucleotide (flap) at the 3' end. However, PcrA did not detectably bind to partial duplexes with folded regions adjacent to a 6 nt long 3' single-stranded tail (with or without a 1 nt flap at the 5' end). PcrA showed moderate helicase activity with partially double-stranded DNAs containing 3' or 5' single-stranded oligo dT tails, the 3'5' helicase activity being more efficient than its 5'3' helicase activity. Interestingly, PcrA showed maximal helicase activity with substrates containing folded structures and 5' single-stranded tails, suggesting that its 5'3' helicase activity is greatly stimulated in the presence of specific structures. However, the 3'5' helicase activity of PcrA did not appear to be affected by the presence of folded substrates containing 3' single-stranded tails. Our data indicate that PcrA may recognize DNA substrates with specific structures in vivo and its 5'3' and 3'5' helicase activities may be involved in distinct cellular processes.
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