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J Biol Chem. 2016 Jun 10;291(24):12467-80. doi: 10.1074/jbc.M116.719591. Epub 2016 Apr 4.

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase.

Author information

  • 1From the Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
  • 2the Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306.
  • 3the Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798, and.
  • 4the National High Magnetic Field Laboratory, Tallahassee, Florida 32310.
  • 5the Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, the National High Magnetic Field Laboratory, Tallahassee, Florida 32310.
  • 6the Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798, and michael_trakselis@baylor.edu.

Abstract

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.

KEYWORDS:

ATPases associated with diverse cellular activities (AAA); DNA helicase; DNA replication; DNA-protein interaction; HDX-MS; MCM; archaea; steric exclusion wrapping

PMID:
27044751
PMCID:
PMC4933441
[Available on 2017-06-10]
DOI:
10.1074/jbc.M116.719591
[PubMed - indexed for MEDLINE]
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