DNA-BINDING OF CPF1 IS REQUIRED FOR OPTIMAL CENTROMERE FUNCTION BUT NOT FOR MAINTAINING METHIONINE PROTOTROPHY IN YEAST

The centromere and promoter factor 1 (CPF1) binds specifically in vitro and in vivo to an octanucleotide (RTCACRTG). This sequence is found in the centromere DNA element I (CDEI) of yeast centromeres and upstream from a number of transcription units including MET25, GAL2 and TRP1. Inactivation of the CPF1 gene results in three phenotypes; slow growth, a partial loss of centromere function and methionine auxotrophy. These phenotypes correlate well with the known binding sites for CPF1 and have led to the suggestion that CPF1 functions as a kinetochore protein at centromeres and as a transcriptional activator at promoters such as MET25. By analysing transcription from the MET25, GAL2, and TRP1 genes in cpf1 strains, we demonstrate that CPF1 plays no direct role in their transcriptional regulation. Further evidence in support of this comes from the analysis of point mutations in the basic region of CPF1 that affect DNA binding. A strain expressing a non-DNA bound form of CPF1 is phenotypically Met+, shows normal growth rate but has sub-optimal centromere function. We conclude that a DNA-bound form of CPF1 is required for the kinetochore function but not for maintaining methionine prototrophy.