Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine

Mark S Dillingham, Dale B Wigley, Martin R Webb

Research output: Contribution to journalArticle (Academic Journal)peer-review

97 Citations (Scopus)

Abstract

Use of the fluorescent base analogue 2-aminopurine has provided a direct demonstration of the translocation of PcrA helicase toward the 5'-end of single-stranded DNA. Single 2-aminopurine bases are introduced into otherwise standard oligonucleotides and produce a fluorescence signal when PcrA reaches their position. We demonstrate that random binding of PcrA to ssDNA is followed by translocation in an ATP-dependent manner toward the 5'-terminus at 80 bases per second at 20 degrees C. The data also provide information on the kinetics of ssDNA binding to the helicase and of the protein dissociation from the 5'-end of ssDNA. A full kinetic model is presented for ATP-dependent DNA translocation by PcrA helicase.
Translated title of the contributionDirect measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine
Original languageEnglish
Pages (from-to)643 - 651
Number of pages9
JournalBiochemistry
Volume41
Issue number2
DOIs
Publication statusPublished - Jan 2002

Bibliographical note

Publisher: American Chemical Society

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