title: MCM ring hexamerization is a prerequisite for DNA-binding.
abstract: The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.
authors.affiliation: Department of Structural Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 311, Memphis, TN 38105, USA.
authors.forename: Clifford A
authors.initials: CA
authors.lastname: Froelich
authors.affiliation: Molecular Interaction Analysis Shared Resource, St Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 311, Memphis, TN 38105, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nourse
authors.affiliation: Department of Structural Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 311, Memphis, TN 38105, USA eric.enemark@stjude.org.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Enemark
mesh_terms: D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4 / Q000235:genetics; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D020027:Pyrococcus furiosus
chemical_list: D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C516793:Mcm4 protein, mouse; D064148:Minichromosome Maintenance Complex Component 4; D064110:Minichromosome Maintenance Proteins