Abstract
In all dividing eukaryotic cells, the mitotic spindle (composed primarily of microtubules) must interact with chromosomes through a complex protein assembly called the kinetochore. In Saccharomyces cerevisiae, the Dam1-DASH complex plays an important role in promoting attachment between the kinetochore and the mitotic spindle. It also actively participates in the physical separation of sister chromatids in anaphase. Understanding the biochemical mechanisms used by Dam1-DASH has been facilitated by bacterial co-expression of the ten Dam1-DASH genes, which results in the production of a heterodecameric protein complex that can be studied in vitro. However, individual protein subunits are not soluble when expressed in E. coli, thus precluding analysis of the nature of the interaction between subunits and an examination of the assembly of the functional complex. In this paper, we describe the expression, solubilization, purification and refolding of Dad1p, one of the Dam1-DASH complex subunits. In addition, we show that Dad1p, when isolated in this manner forms dimers and/or tetramers, dependent upon protein concentration. This work provides an important tool for studying the Dam1-DASH complex that was previously unavailable, and provides an avenue of investigation for understanding how the individual heterodecamers associate with each other to facilitate chromosome segregation.
Highlights
Cell division, in general, and chromosome segregation, in particular, are areas of immense concern to both the cell and the cell biologist
In an effort to better understand the contribution of this protein to Dam1DASH kinetochore function, we show that isolated Dad1p is likely a tetramer or dimer
All yeasts appear to have Dam1-DASH complex, regardless of whether they use a specific sequence to identify the location of the kinetochore, as in S. cerevisiae, or if they have a regional kinetochore assembly process, as in S. pombe [12]
Summary
In general, and chromosome segregation, in particular, are areas of immense concern to both the cell and the cell biologist. Understanding how these processes have evolved into reliable cellular functions is a goal of basic research and of interest to those pursuing drug development and design. The best studied is the yeast Saccharomyces cerevisiae [1]. In this organism, there are believed to be at least 65 different proteins that work together to allow the kinetochore to function properly. An important goal in furthering our understanding of mitosis is to understand the contribution and regulation of each of these proteins
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