Subunit affinities and stoichiometries of the human papillomavirus type 11 E1:E2:DNA complex.
The association between the papillomavirus E1 and E2 proteins is an important regulatory interaction, imparting coordinated control of viral transcription and replication. Using fluorescence polarization, we have characterized the interactions between HPV-11 E1, HPV-11 E2, and DNA in solution at equilibrium. For these studies, two double-stranded fluorescein-labeled oligonucleotides were prepared. The first fluorescent oligonucleotide, designated Fl-E2BS and containing a single E2 binding-site palindrome (ACCGN6CGGT), was used to determine the affinity of E2 for its DNA binding site. HPV-11 E2 bound Fl-E2BS with an apparent Kd of 0.84 nM. Binding was saturable and consistent with a single class of noninteracting sites. The second oligonucleotide, designated Fl-E1E2BS, contained both E1 and E2 sites in sequence derived directly from the HPV-11 origin of replication. Under titration conditions identical to those used for Fl-E2BS, the E2 protein exhibited reduced affinity for Fl-E1E2BS (Kd > 100 nM). E1 binding to Fl-E1E2BS was of very low affinity. Addition of excess HPV-11 E1 to Fl-E1E2BS lowered the dissociation constant for the E2:Fl-E1E2BS interaction to 2 nM. This effect was not dependent upon ATP or magnesium ion. Fluorescence polarization and other data suggest formation of a complex containing six E1 molecules and a single dimer of E2 bound to a single Fl-E1E2BS oligonucleotide; E2 dissociation from the final complex did not occur. In summary, physical interaction between E1 and E2 increases the DNA binding affinity of each. The role of this energy coupling may be to promote origin-specific binding of both E1 and E2 to DNA.
Chao
S F
SF
Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rocque
W J
WJ
Daniel
S
S
Czyzyk
L E
LE
Phelps
W C
WC
Alexander
K A
KA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D027383:Papillomaviridae / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; C117355:E2 protein, Human papillomavirus type 11; D005452:Fluoresceins; D046911:Macromolecular Substances; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D008274:Magnesium