Centromeres are stress-induced fragile sites

Abstract

Centromeres are unique loci on eukaryotic chromosomes and are complexed with centromere-specific histone H3 molecules (CENP-A in mammals, Cse4 in yeast). The centromere provides the binding site for the kinetochore that captures microtubules and provides the mechanical linkage required for chromosome segregation. Centromeres encounter fluctuations in force as chromosomes jockey for position on the metaphase spindle. We have developed biological assays to examine the response of centromeres to high force. Torsional stress is induced on covalently closed DNA circles from supercoiling. Plasmid-borne centromeres with single-nucleotide inactivating mutations exhibit a high conversion frequency to plasmid dimer species. Conversion to dimers is dependent on the activity of the Rad1 single-strand endonuclease, indicative of unwinding a region of the centromere sequence in the absence of a functional kinetochore. To determine the region of unwinding, we used conditionally functional dicentric chromosomes to exert tension. Centromere DNA is exquisitely sensitive to cleavage following activation of the dicentric chromosome. Cleavage is dependent on the action of Rad1, highlighting the propensity of centromeres to unwind in response to supercoiling or mechanical stress. These studies provide mechanistic insights into the evolution of AT-rich pericentromere DNA throughout phylogeny and suggest a mechanism for stress-induced error correction at the centromere.

Keywords: CENP-A nucleosome; DSB; Rad1 nuclease; centromere DNA; fragile sites; supercoiling tension.