In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae

Abstract

The autonomously replicating sequence-binding factor 1 (ABF1) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABF1 binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5′-CGTnnnnnnnGA(G/C)-3′. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABF1. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABF1. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABF1 with nearly two-magnitude higher affinity as compared to the hitherto accepted ABF1 consensus sequence. This led us to postulate a strong recognition motive: 5′-TnnCGTnnnnnnTGAT-3′.