Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres

Abstract

Centromeres most likely consist of DNA (CEN DNA) interacting with specific proteins. In Saccharomyces cerevisiae a clear picture has emerged of a 120 bp sequence that is characteristic of CEN DNA. We have investigated the 25 bp centromere DNA element (CDEIII) that represents the right part of a CEN DNA. We showed using a series of mutants generated in vitro that the right most triple A of the consensus sequence TGT.T.TG.. TTCCGAA.....AAA participates in the assembly of a functional centromere and that no further sequences to the right are needed. Distance changes between the centre dyad TTCCGAA and the triple A have two effects: Addition of one base pair leads to a reduction, and addition of two or four base pairs to a loss of centromere function implying a participation of the centre dyad and the triple A region in protein binding. Indeed, a synthetic oligonucleotide of 39 bp containing CDEIII shows specific protein binding.