title: The Smc5-Smc6 heterodimer associates with DNA through several independent binding domains.
abstract: The Smc5-6 complex is required for the maintenance of genome integrity through its functions in DNA repair and chromosome biogenesis. However, the specific mode of action of Smc5 and Smc6 in these processes remains largely unknown. We previously showed that individual components of the Smc5-Smc6 complex bind strongly to DNA as monomers, despite the absence of a canonical DNA-binding domain (DBD) in these proteins. How heterodimerization of Smc5-6 affects its binding to DNA, and which parts of the SMC molecules confer DNA-binding activity is not known at present. To address this knowledge gap, we characterized the functional domains of the Smc5-6 heterodimer and identify two DBDs in each SMC molecule. The first DBD is located within the SMC hinge region and its adjacent coiled-coil arms, while the second is found in the conserved ATPase head domain. These DBDs can independently recapitulate the substrate preference of the full-length Smc5 and Smc6 proteins. We also show that heterodimerization of full-length proteins specifically increases the affinity of the resulting complex for double-stranded DNA substrates. Collectively, our findings provide critical insights into the structural requirements for effective binding of the Smc5-6 complex to DNA repair substrates in vitro and in live cells.
authors.affiliation: Institute for Research in Immunology and Cancer, and Département de Pathologie et biologie cellulaire, Université de Montréal P.O. Box 6128, Succursale Centre-Ville Montréal, QC, H3C 3J7, Canada.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Roy
authors.affiliation: Institute for Research in Immunology and Cancer, and Département de Pathologie et biologie cellulaire, Université de Montréal P.O. Box 6128, Succursale Centre-Ville Montréal, QC, H3C 3J7, Canada.
authors.forename: Thillaivillalan
authors.initials: T
authors.lastname: Dhanaraman
authors.affiliation: Institute for Research in Immunology and Cancer, and Département de Pathologie et biologie cellulaire, Université de Montréal P.O. Box 6128, Succursale Centre-Ville Montréal, QC, H3C 3J7, Canada.
authors.forename: Damien
authors.initials: D
authors.lastname: D'Amours
mesh_terms: D000255:Adenosine Triphosphate; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D013379:Substrate Specificity
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA