Nucleic Acids Research, 1991, Vol. 19, No. 14 3913-3919
© 1991
MOLECULAR BIOLOGY |
Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein
Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX and Ludwig Institute for Cancer Research, St Mary's Hospital Medical School Norfolk Place, London W2 1PG, UK
* To whom correspondence should be addressed
Received April 25, 1991. Revised June 18, 1991. Accepted June 18, 1991.
Using a binding site selection procedure, we have found that sequence-specific DNA-binding by the mouse c-myb protein involves recognition of nucleotides outside of the previously identified hexanucleotide motif. Ollgonucleotides containing a random nucleotide core were immunoprecipitated in association with c-Myb, amplified by the Polymerase Chain Reaction and cloned in plasmids prior to sequencing. By alignment of sequences it was apparent that additional preferences existed at each of three bases immediately 5' of the hexanucleotide consensus, allowing an extension of the preferred binding site to YGRCVGTTR. The contributions of these 5' nucleotides to binding affinity was established in bandshift analyses with oligonucleotides containing single base substitutions; in particular, it was found that replacement of the preferred guanine at position - 2 with any other base greatly reduced c-Myb binding. We found that the protein encoded by the related B-myb gene bound the preferred c-Myb site with similar affinity; however, B-Myb and c-Myb showed distinct preferences for the identity of the nucleotide at position -1 relative to the hexanucleotide consensus. This study demonstrates that the c-Myb DNA-binding site is more extensive than recognised hitherto and points to similar but distinct nucleotide preferences in recognition of DNA by related Myb proteins.
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