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

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit p rotein complex that binds to the essential CDEIII element in CEN DNA. The f our 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, Asse mbly of CBF3-CEN DNA complexes was performed by combining purified CBF3 pro teins with a DNA fragment that includes the CEN region from yeast chromosom e III. Atomic force microscopy images showed DNA molecules with attached gl obular bodies. The contour length of the DNA containing the complex is appr oximate to 9% shorter than the DNA alone, suggesting some winding of DNA wi thin the complex, The measured location of the single binding site indicate s that the complex is located asymmetrically to the right of CDEIII extendi ng away from CDEI and CDEII, which is consistent with previous data. The CE N DNA is bent approximate to 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three t o four DNA arms,,vith molecular volumes approximately three times the mean volumes of two-armed complexes, These multi-armed complexes indicate that C BF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.