Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5-6 Subcomplex and the Hinge Regions of Smc5 and Smc6

Xinyuan Duan,Yan Yang,Yu-Hung Chen,Jacqueline Arenz,Gurdish K Rangi,Xiaolan Zhao,Hong Ye

doi:10.1074/jbc.m809139200

Abstract

The evolutionarily conserved structural maintenance of chromosome (SMC) proteins forms the core structures of three multisubunit complexes as follows: cohesin, condensin, and the Smc5/6 complex. These complexes play crucial roles in different aspects of chromosomal organization, duplication, and segregation. Although the architectures of cohesin and condensin are better understood, that of the more recently identified Smc5/6 complex remains to be elucidated. We have previously shown that the Smc5/6 complex of Saccharomyces cerevisiae contains Smc5, Smc6, and six non-SMC elements (Nse1-6). In this study, we investigated the architecture of the budding yeast Smc5/6 complex employing the yeast two-hybrid assay as well as in vitro biochemical approaches using purified recombinant proteins. These analyses revealed that Smc5 and Smc6 associate with each other at their hinge regions and constitute the backbone of the complex, whereas the Nse1-6 subunits form three distinct subcomplexes/entities that interact with different regions of Smc5 and Smc6. The Nse1, -3, and -4 subunits form a stable subcomplex that binds to the head and the adjacent coiled-coil region of Smc5. Nse2 binds to the middle of the coiled-coil region of Smc5. Nse5 and Nse6 interact with each other and, as a heterodimer, bind to the hinge regions of Smc5 and Smc6. These findings provide new insights into the structures of the Smc5/6 complex and lay the foundation for further investigations into the mechanism of its functions.

Highlights

These three structural maintenance of chromosome (SMC) complexes play distinct roles in chromosomal organization and functions
Pairwise Two-hybrid Tests between Subunits of the Smc5/6 Complex Reveals Three NSE Entities That Bind to Smc5—As an initial step to study the architecture of the budding yeast Smc5/6 complex, we systematically examined the interactions between all the subunits of the Smc5/6 complex
Each of the eight subunits of the budding yeast Smc5/6 complex was cloned into two-hybrid vectors allowing them to be fused with either a GAD (Gal4 activation domain) or a GBD (Gal4 DNA-binding domain)

Summary

EXPERIMENTAL PROCEDURES

Plasmids Constructs—The hinge, head, and coiled-coil regions of Smc and Smc were determined using the secondary structure prediction program PredictProtein (PHD) and the homologous alignment of these proteins with other SMC proteins. 50 –100 ␮g of testing proteins were added to the beads and incubated for 30 min at 4 °C with rotation. Two-hybrid strain pJ69-4a was transformed with GAD vector (pOAD) or GAD fusion plasmids; transformants were selected and grown on SCLEU medium. Two-hybrid strain pJ69-4␣ was transformed with GBD vector (pOBD) or GBD fusion plasmids and was selected and grown on SC-TRP medium. Both pOAD and pOBD are CEN plasmids and present 1–3 copies/cell. The resulting transformants were mated, and the diploid cells containing both the GAD and GBD constructs were selected on SC-TRP-LEU plates. Each construct was tested with vector alone as a control

RESULTS

Amino acid position

DISCUSSION

IW E W E