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Nucleic Acids Res. 2019 Aug 22;47(14):7494-7501. doi: 10.1093/nar/gkz541.

Direct quantification of the translocation activities of Saccharomyces cerevisiae Pif1 helicase.

Author information

1
Mechanobiology Institute, National University of Singapore, Singapore 117411.
2
Centre for Bioimaging Sciences, National University of Singapore, Singapore 117557.
3
Department of Physics, National University of Singapore, Singapore 117542.
4
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Science, Arkansas 72205, USA.
5
School of Biological Sciences, Nanyang Technology University, Singapore 637551.

Abstract

Saccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 μM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a 'mobile' manner.

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