Neocentromeres form efficiently at multiple possible loci in Candida albicans

Area of Science:

• * Molecular Biology
• * Genetics
• * Mycology

Background:

• * Centromeres are essential for chromosome stability, typically featuring repetitive DNA and pericentric heterochromatin in most eukaryotes.
• * Neocentromeres can form at new locations, as DNA sequence is not strictly required for kinetochore assembly.
• * Candida albicans, a human fungal pathogen, possesses small regional centromeres without pericentric heterochromatin.

Purpose of the Study:

• * To investigate the properties and formation of neocentromeres in Candida albicans.
• * To functionally delimit centromere DNA on Chromosome 5 (CEN5) and assess neocentromere assembly.
• * To analyze neocentromere movement and its dependence on wild-type centromere DNA.

Main Methods:

• * Functional deletion of CEN5 DNA and replacement with marker genes (e.g., URA3).
• * Assessment of chromosome stability and growth in transformants lacking CEN5 DNA.
• * Massively parallel sequencing of DNA isolated by CENP-A(Cse4p) chromatin immunoprecipitation (ChIP) to map neocentromere locations.

Main Results:

• * Neocentromeres assembled efficiently on homologs lacking CEN5 DNA, enabling stable retention of Chromosome 5.
• * Neocentromere function was independent of the presence of wild-type CEN5 DNA.
• * Two classes of neocentromeres were identified: proximal (adjacent to deleted CEN5) and distal (200-450 kb away), with proximal neocentromeres causing URA3 gene silencing.

Conclusions:

• * Functional neocentromeres form readily in C. albicans at loci with low gene density and flanking repeats.
• * Neocentromeres can move locally, evidenced by URA3 silencing, or relocate to different chromosomal regions.
• * This system allows for mechanistic studies of regional centromere assembly and maintenance in C. albicans.