Probing the Saccharomyces cerevisiae centromeric DNA ( CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Abstract

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is Ϸ9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent Ϸ55°at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.Accurate chromosome segregation in mitosis and meiosis depends on the correct assembly of kinetochores on centromeric DNA (CEN DNA). During cell division, these structures attach chromosomes to the microtubules of the mitotic spindle and move the replicated chromosomes to opposing spindle poles.The minimal functional centromere in the budding yeast Saccharomyces cerevisiae contains a 125-base-pair sequence (CEN) present once on each chromosome, which is organized into three domains: CDEI, CDEII, and CDEIII ( In this paper, we describe the use of atomic force microscopy (AFM; also called scanning force microscopy) to probe the organization of the CBF3-CEN DNA complexes. We find that binding of the CBF3 proteins is associated with DNA shortening and bending. The location of the center of the CBF3 complex relative to the CDEIII site was found to be quite asymmetric, corroborating previous findings from DNase protection and DNA-protein crosslinking studies (2, 3). Additionally, we encountered evidence of joining or pairing of CEN DNA molecules by the CBF3 proteins. AFM has been successfully used to image biological samples in air or in liquid (4-8), and our findings further demonstrate that this imaging method is a powerful tool for the study of macromolecular assemblies and their interactions (9-16). MATERIALS AND METHODSPreparation of CBF3 Proteins. Cbf3a, Cbf3b, and Cbf3c͞d complex proteins tagged with 6-10 histidine residues were overexpressed and purified from Pichia pastoris, Escherichia coli, and S. cerevisiae, respectively, according to the procedure described in ref. 23.Preparation of CEN DNA Fragments and pUC19 DNA Beads. A linear DNA restriction fragment con...

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