The Requirement for the Dam1 Complex Is Dependent upon the Number of Kinetochore Proteins and Microtubules

Abstract

Summary The Dam1 complex attaches the kinetochore to spindle microtubules and is a processivity factor in vitro [1, 2]. In Saccharomyces cerevisiae, which has point centromeres that attach to a single microtubule, deletion of any Dam1 complex member results in chromosome segregation failures and cell death [3–5]. In Schizosaccharomyces pombe, which has epigenetically-defined regional centromeres that each attach to 3–5 kinetochore microtubules, Dam1 complex homologs are not essential [6]. To ask why the complex is essential in some organisms and not in others, we used Candida albicans, a multimorphic yeast with regional centromeres that attach to a single microtubule [7]. Interestingly, the Dam1 complex was essential in C. albicans, suggesting that the number of microtubules per centromere is critical for its requirement. Importantly, by increasing CENP-A expression levels, more kinetochore proteins and microtubules were recruited to the centromeres, which remained fully functional. Furthermore, Dam1 complex members became less crucial for growth in cells with extra kinetochore proteins and microtubules. Thus, the requirement for the Dam1 complex is not due to the DNA-specific nature of point centromeres. Rather, the Dam1 complex is less critical when chromosomes have multiple kinetochore complexes and microtubules per centromere, implying that it functions as a processivity factor in vivo as well as in vitro.

Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi

“…The reason for this unusual pattern of centromeric chromatin seen in vivo is still unknown, but it may be attributable to replacement of histone H3 by CENP-A. CENP-A molecules have been shown to replace histone H3 molecules either partially or fully in centromeric chromatin in different fungi (17,80,110,123). Studies in S. pombe have suggested that the unusual smeary pattern of partial MNase digestion could be due to protection provided by an intact kinetochore.…”

“…In S. cerevisiae, CENP-A/Cse4 proteolysis, which is mediated by Psh1, an E3 ubiquitin ligase, restricts its localization primarily to the centromere (30,56,102). Overexpression of CENP-A/CaCse4 results in recruitment of more CENP-A/CaCse4 molecules along with other kinetochore proteins such as Mtw1 at the centromere in C. albicans (17,106). Posttranslational modifications associated with the N-terminal histone tail are related to different functional states of chromatin-like repression (silencing) or activation of transcription.…”

Divalent Cations Affect the Stability and Structure of Dad2p, a Subunit of the Candida Albicans Kinetochore Dam1 Complex

“…microtubule: 1 kinetochore. This is the case in S. cerevisiae (Cheeseman et al, 2001;Janke, Ortiz, Tanaka, Lechner, & Schiebel, 2002;Li, Li, & Elledge, 2005) and C. albicans (Burrack, Applen, & Berman, 2011;Thakur & Sanyal, 2011), but not so for S. pombe (Sanchez-Perez et al, 2005). This difference has been postulated to reflect the more stringent requirement for maintaining connection with the depolymerizing microtubule when a single point of attachment is present; thus, under these circumstances, the ring may provide necessary stability (Burrack et al, 2011;Thakur & Sanyal, 2011).…”

“…This is the case in S. cerevisiae (Cheeseman et al, 2001;Janke, Ortiz, Tanaka, Lechner, & Schiebel, 2002;Li, Li, & Elledge, 2005) and C. albicans (Burrack, Applen, & Berman, 2011;Thakur & Sanyal, 2011), but not so for S. pombe (Sanchez-Perez et al, 2005). This difference has been postulated to reflect the more stringent requirement for maintaining connection with the depolymerizing microtubule when a single point of attachment is present; thus, under these circumstances, the ring may provide necessary stability (Burrack et al, 2011;Thakur & Sanyal, 2011). Outside of the yeasts, sequence-based homologues of the Dam1 complex proteins are not identifiable, though a variety of evidence suggests that the functional homologue in metazoans may be the Ska complex (Gaitanos et al, 2009;Guimaraes & Deluca, 2009;Hanisch, Sillje, & Nigg, 2006;Jeyaprakash et al, 2012;Welburn et al, 2009).…”

Plo1 phosphorylates Dam1 to promote chromosome bi-orientation in fission yeast

“…The DASH complex is conserved in other fungi but not in metazoa. The requirement for DASH is related to the number of microtubule-binding sites per kinetochore (Burrack et al, 2011;Thakur and Sanyal, 2011). Although the DASH complex is essential for viability in budding yeast, where kinetochores bind to a single microtubule, it is not required for viability in fission yeast, where kinetochores bind to between two and four microtubules (Liu et al, 2005;Sanchez-Perez et al, 2005).…”