Luo, Bo
Mapping of sequence specific DNA- protein interactions: a versatile, quantitative method and its applications to transcription factor XF1.

Order No.9608533

Mapping the consensus sequence of DNA binding proteins has been greatly accelerated by methods that use in vitro selection of high affinity sequences from a library of random DNA molecules, followed by PCR-amplification and sequence analysis. However, these methods lose other valuable information because they use repetitive cycles of selection and amplification.

We have developed a method that overcomes this limitation, not only defining the consensus sequence, but also quantitating the effect on DNA-protein affinity of replacing each base in the recognition domain with every other base. The features of this method are: (1) Instead of synthesizing one oligonucleotide population containing a long randomized domain, we synthesize several oligonucleotide populations, each randomized at two positions. Because only a few species are present in each population, the concentration of each is sufficient to saturate the DNA-binding protein. Consequently, the abundance of each protein-bound oligonucleotide accurately reflects its binding affinity. (2) Because only a few species are represented in each oligonucleotide population, a single round of selection and amplification generates sufficient material for sequencing. This avoids biasing the population of protein-bound oligonucleotides toward high affinity species. Consequently, the abundances of oligonucleotides determined by sequence analysis accurately reflects their binding affinities. (3) We developed data collection and analysis procedures that eliminate artifacts, and yield accurate measures of: (a) the selectivity of the protein for each base at each position within the recognition domain (normalized relative selectivity), (b) the contributions of individual sites within the recognition domain to the binding affinity (selectivity variance), (c) the relative affinity of a particular sequence for the DNA-binding protein (global selectivity). (4) We developed a procedure for deducing aspects of the matrix of hydrogen bonds involved in DNA-protein interactions.

This method was first developed and applied to the nuclear protein XF1, which binds to xenobiotic responsive elements, that class of elements through which the liganded Ah receptor activates transcription of the CYP1A1 gene. We confirmed results by (1) cloning and sequencing individual XF1-bound oligonucleotides, and (2) competition EMSA analysis of oligonucleotides designed on the basis of in vitro selection results. Source: DAI, 56, no. 11B, (1996): 5943


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