The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function.Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIH, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.For faithful transmission of genetic material in eucaryotes, two main processes are responsible: mitosis and meiosis. During these processes, the duplicated chromosomes become attached to the spindle fibers and subsequently segregate in an orderly fashion to mother and daughter cells. Attachment of the chromosomes to the spindle occurs via the kinetochore at a specific chromosomal segment termed the centromere. The centromere DNA (CEN DNA) is bound by specific centromere proteins and is thought to be the organizing center for the kinetochore. So far, only for the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe has it been possible to isolate centromere DNA (13-15, 39, 40). In the budding yeast S. cerevisiae, all meiotic and mitotic centromere functions required in cis are contained within a 125-bp CEN DNA fragment (16). This fragment comprises three centromere DNA elements (CDEI, CDEII, and CDEIII) conserved throughout the 13 CEN DNAs that have been isolated thus far (19, 27, 29, 30a) (Fig. 1). Chromatin analysis has uncovered a specific centromere chromatin structure (5). A detailed analysis revealed a region of approximately 160 bp, including CDEI, CDEII, and CDEIII, which is protected against nuclease digestion (20). In vivo footprint analysis identified specific G nucleotides within CDEI and CDEIII as protected against methylation, indicating tight DNA-protein interactions at these sites (36, 50).The first evidence that the CDEI sequence RTCACRTG (R = purine) may act as a protein-binding site was presented by Bram and Kornberg, who identified an activity in partly purified yeast protein extracts which bound not only to the CDEI sequence in the centromere, but also to a CDEI sequence found in the GAL2 promoter (6). Subsequently, other groups purified this protein, referred to variously as CPF1 (centromere and promoter factor), CP1 (centromere protein), and CBF1 (centromere-binding factor), and cloned * Corr...