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Illuminating the elementary steps of helicase translocation and unwinding via single molecule analysis
Author(s) -
Ha Taekjip
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.99.2
Subject(s) - helicase , chromosomal translocation , dna , atp hydrolysis , biophysics , chemistry , biology , crystallography , biochemistry , gene , enzyme , rna , atpase
The step sizes of DNA translocation and unwinding by helicases have been a subject of significant biophysics interest for some time. In principle, up to 10 base pairs (bp) of DNA can be unwound using the energy liberated by one ATP hydrolyzed. Translocation on single stranded DNA (ssDNA)has even less constraints. Past structural studies suggested the smallest step size of 1 nt translocation per ATP but functional studies suggested larger physical or kinetic step sizes ranging from 4 to 23 nt/bp translocation/unwinding. We have used single molecule fluorescence techniques to deduce the ssDNA translocation step size of PcrA helicase and unwinding step size of hepatitus C virus NS3 helicase. The former was made possible by the highly regular cycles of repetitive ssDNA looping coupled with PcrA translocation. In NS3, we found kinetic evidence that each 3 bp unwinding step requires approximately 3 hidden steps, presumably due to 1 ATP hydrolysis each. Our results reconcile the apparent discrepancies between the structural and biochemical data, and may prove general for many other helicases.