Amidst Multiple Binding Orientations on Fork DNA, Saccharolobus MCM Helicase Proceeds N-first for Unwinding

Elife. 2019 Oct 29;8:e46096. doi: 10.7554/eLife.46096.

Abstract

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.

Keywords: MCM; biochemistry; chemical biology; helicase; translocation; unwinding.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Helicases / metabolism*
  • DNA Replication*
  • DNA, Archaeal / metabolism*
  • Minichromosome Maintenance Proteins / metabolism*
  • Protein Binding
  • Protein Domains
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / metabolism*

Substances

  • DNA, Archaeal
  • DNA Helicases
  • Minichromosome Maintenance Proteins