title: 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.
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.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
authors.forename: Brian W
authors.initials: BW
authors.lastname: Graham
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306.
authors.forename: Yeqing
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798.
authors.forename: Katie L
authors.initials: KL
authors.lastname: Dodge
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798.
authors.forename: Carly T
authors.initials: CT
authors.lastname: Thaxton
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798.
authors.forename: Danae
authors.initials: D
authors.lastname: Olaso
authors.affiliation: National High Magnetic Field Laboratory, Tallahassee, Florida 32310.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Young
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306; National High Magnetic Field Laboratory, Tallahassee, Florida 32310.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798. Electronic address: michael_trakselis@baylor.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Trakselis
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D017357:Cyclotrons; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods*; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D004265:DNA Helicases; D064110:Minichromosome Maintenance Proteins
keywords: ATPases associated with diverse cellular activities (AAA); DNA helicase; DNA replication; DNA-protein interaction; HDX-MS; MCM; archaea; steric exclusion wrapping