Purification of a yeast centromere-binding protein that is able to distinguish single base-pair mutations in its recognition site.

Abstract

A centromere-specific DNA-binding protein has been purified to homogeneity by a combination of conventional and sequence-affinity chromatography from the yeast Saccharomyces cerevisiae. This protein (designated CBP-I) has an apparent molecular weight of 16,000. It binds specifically to the CDEI (centromere DNA element I) region of yeast centromere DNA, as shown by the electrophoretic mobility retardation assay and DNase I protection analysis, but does not bind specifically to other regions of yeast centromere DNA such as CDEII and CDEIII. The relative binding affinity of purified CBP-I to five different point mutations of CDEI correlates directly with the previously determined ability of each point mutation to convey centromere function in a mitotic chromosome segregation assay

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Although most eukaryotic centromeres are maintained epigenetically (Black et al 2010;Henikoff and Furuyama 2010), yeast centromeres are genetically specified by DNA sequence. The CDEI consensus sequence (PuTCACPuTG) binds to the helix-loop-helix protein Cbf1 (Cai and Davis 1989;Baker and Masison 1990;Cai and Davis 1990), a transcription factor that also binds to other elements throughout the genome. CDE1 and Cbf1 contribute to kinetochore function but are not essential.…”

Chromatin Proteins Are Determinants of Centromere Function

“…A subset of these proteins bind specifically to the CDE elements present in centromeric DNA. For example, CDEI recruits the Cbf1 protein to mark the left boundary of the protected core domain (Saunders et al 1988;Bram and Kornberg 1987;Baker et al 1989;Cai and Davis 1989;Jiang and Philippsen 1989). Chromatin digestion of cbf1Δ cells results in a slightly reduced area of nuclease protection of the core centromere, and both CDEIΔ and cbf1 Δ cells display only minor defects in chromosome segregation (Cai and Davis 1990).…”

Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.

“…In its stability and DNA-binding properties, CP1 is similar to a protein in HeLa extracts which appears to be the adenovirus major late transcription factor (named MLTF or USF) (6,12,13,42). Cai and Davis have purified a CDEI-binding protein from yeast cells which they call CBP-I (9). CBP-I has a molecular weight of 16,000 but has DNAbinding properties identical to those of CP1.…”