Purification of yeast replication origin binding proteins

Abstract

Abstract This chapter provides practical advice on the biochemical purification of proteins that bind to replication origins in the budding yeast Saccharomyces cerevisiae. Genetic and biochemical analysis in budding yeast has facilitated much of our current understanding about the initiation of replication of chromosomal DNA in eukaryotic cells, in large part because only in this organism have the DNA sequences that serve as replication origins been identified and characterized in detail. Replication origins in S. cerevisiae consist of discrete 100--300 bp sequences termed autonomously replicating (ARS) sequences that are both necessary and sufficient for replication of plasmid DNA. At least a subset of ARSs function as origins of replication in their native chromosomal context. The combined power of biochemical and genetic approaches available in yeast has led to the identification of proteins required for initiation of DNA replication that interact, either directly or indirectly, with ARS sequences. Furthermore, recent progress has demonstrated the periodic assembly of complexes involving these proteins at replication origins during the cell cycle and that this process is regulated by cyclin dependent kinases (CDKs). The goal of this chapter is to provide protocols and necessary information for the purification of three origin interacting factors: ORC, Abflp, and Cdc46p, a member of the Mcm family of proteins. Recent evidence also implicates Cdc6p, Dbf4p, and Cdc45p as origin inter acting factors. The characteristics of Cdc6p, Dbf4p, and Cdc45p will be briefly reviewed, however no protocols are yet available for the purification of these proteins from yeast. For more comprehensive discussion of ARS sequence characteristics, ARS binding proteins, and cell cycle regulation of initiation the reader is directed to a number of recent reviews (1-6).