4.8 Article

Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB

Journal

NUCLEIC ACIDS RESEARCH
Volume 46, Issue 16, Pages 8500-8515

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/nar/gky647

Keywords

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Funding

  1. European Research Council [MAGREPS 267 862]
  2. Juan de la Cierva Program MICINN-JDC
  3. Science and Engineering Research Board grant [EMR/2016/003910]

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Most RecQ DNA helicases share a conserved domain arrangement thatmediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a 'Helicase and RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, 'slow' and 'normal', as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third 'fast' unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.

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