Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.
To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.
van Leeuwen
H C
HC
Laboratory for Physiological Chemistry, Utrecht University, Stratenum, The Netherlands.
Strating
M J
MJ
Cox
M
M
Kaptein
R
R
van der Vliet
P C
PC
D000256:Adenoviridae / Q000502:physiology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027702:Organic Cation Transporter 1; D050809:POU Domain Factors; D017354:Point Mutation; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D027702:Organic Cation Transporter 1; D050809:POU Domain Factors; D014157:Transcription Factors; D004247:DNA