Identifying Functional Domains in Subunits of Structural Maintenance of Chromosomes (SMC) Complexes by Transposon Mutagenesis Screen in Yeast.

Abstract

Structural maintenance of chromosomes (SMC) complexes mediate higher order chromosome structures. Eukaryotic cells contain three distinct SMC complexes called cohesin, condensin, and SMC5/6, which share the same basic architecture. The core of SMC complexes contains a heterodimer of SMC proteins, a kleisin subunit, and a set of regulatory proteins that contain HEAT and Armadillo (ARM) repeat protein-protein interaction motifs. A major challenge in studying SMC proteins and their auxiliary factors is identifying their functional domains. Bioinformatics is not an efficient way to achieve this goal because of the absence of defined sequence and structural motifs. Functional domains can be identified experimentally by performing a genetic screen and isolating functional mutants. While there are several strategies to conduct a screen, the quaternary structure of SMCs makes them excellent candidates to transposon-based random insertion mutagenesis, followed by selection of dominant negative mutants. In this chapter we list the advantages of this approach in the context of SMC complexes. We provide a detailed protocol for performing the screen in S. cerevisiae and use data from our recently reported screen on the ARM repeat protein, Scc4, to demonstrate the key steps in the protocol.