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Proc Natl Acad Sci U S A. 2016 May 31;113(22):6194-9. doi: 10.1073/pnas.1602878113. Epub 2016 May 16.

Chemo-mechanical pushing of proteins along single-stranded DNA.

Author information

1
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
2
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110 lohman@wustl.edu.

Abstract

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

KEYWORDS:

DNA motors; SF1 translocases; SSB proteins; dynamics

PMID:
27185951
PMCID:
PMC4896671
DOI:
10.1073/pnas.1602878113
[Indexed for MEDLINE]
Free PMC Article

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