title: Interaction mapping between Saccharomyces cerevisiae Smc5 and SUMO E3 ligase Mms21.
abstract: The multisubunit Smc5-Smc6 holocomplex (Smc5/6) plays a critical role in chromosome stability maintenance, DNA replication, homologous recombination, and double-stranded DNA damage repair. Smc5 and Smc6 form the core of the holocomplex, along with six non-SMC elements, for which most functions are not yet understood. Mms21 (Nse2), the relatively well-studied subunit in Smc5/6, contains a SP-like-RING finger motif on the C-terminus and was identified as a SUMO E3 ligase. Deletion of Mms21 is lethal; however, while deficient in DNA damage repair, SUMO ligase mutants remain viable. These functions of Mms21 in Smc5/6 are hard to address without understanding the interaction between Smc5 and Mms21. Previously, we systematically examined the architecture of Saccharomyces cerevisiae Smc5/6 and, using yeast two-hybrid methods, found that Mms21 interacts with the coiled-coil of Smc5. Later, crystallographic studies revealed the molecular arrangement of Mms21 with Smc5/6. For this study, we use a combination of limited proteolysis, mass spectrometry, and N-terminal sequencing to precisely define the interaction region of Smc5 with Mms21. In addition, using isothermal titration calorimetry, we find that Mms21 interacts with Smc5 in a 1:1 ratio with a K(d) of 0.68 μM. This combination of methods would be useful in examining the structure of any large multiprotein complex.
authors: James Graham Brown Cancer Center, University of Louisville, 505 South Hancock Street, Kentucky 40202, USA.
authors: Xinyuan
authors: X
authors: Duan
authors: William B
authors: WB
authors: Holmes
authors: Hong
authors: H
authors: Ye
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D025941:Protein Interaction Mapping / Q000379:methods; D025842:SUMO-1 Protein / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein
chemical_list: D018797:Cell Cycle Proteins; C500948:Mms21 protein, S cerevisiae; C486627:SMC5 protein, S cerevisiae; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins