Functional plasticity in chromosome-microtubule coupling on the evolutionary time scale.

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• The Dam1 complex is vital for accurate chromosome segregation during mitosis in fungi.
• Dad2 is a key subunit of the Dam1 complex, implicated in its function.
• Centromere structure varies significantly across fungal species, from point centromeres to regional centromeres.

Purpose of the Study:

• To investigate the functional significance of a conserved Dad2 signature sequence (DSS) within the Dam1 complex.
• To determine the role of a specific arginine residue (R126) within the DSS in relation to centromere structure.
• To understand how centromere length influences the essentiality of the Dad2 DSS for mitotic progression.

Main Methods:

• Amino acid sequence analysis to identify conserved regions in Dad2.
• Site-directed mutagenesis to create Dad2 variants (Dad2ΔDSS, Dad2R126A).
• Purification of recombinant Dam1 complexes and in vitro binding assays with microtubules (MTs).
• Functional analysis of mutant strains in Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans to assess viability and chromosome biorientation.

Main Results:

• A conserved 10-amino acid Dad2 Signature Sequence (DSS) was identified in Dad2.
• The arginine residue (R126) within the DSS is essential for viability in Saccharomyces cerevisiae (point centromeres) but not in Candida albicans or Cryptococcus neoformans (regional centromeres).
• Mutant Dam1 complexes lacking the DSS or the critical arginine failed to bind MTs or form rings.
• The requirement for the conserved arginine decreased as centromere length increased.

Conclusions:

• The Dad2 DSS and its conserved arginine are critical for Dam1 complex function, particularly MT binding and ring formation.
• The essentiality of the Dad2 arginine residue is inversely correlated with centromere length, highlighting functional adaptation.
• A model is proposed where regional centromeres accommodate mutations in the Dad2 arginine by enhancing kinetochore-MT interactions, possibly through increased kinetochore protein function.