Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein.
The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.
Sim
Jeonggu
J
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.
Ozgur
Sezgin
S
Lin
Biing Yuan
BY
Yu
Jei-Hwa
JH
Broker
Thomas R
TR
Chow
Louise T
LT
Griffith
Jack
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D019281:Dimerization; D052140:Human papillomavirus 11 / Q000235:genetics*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018741:Replication Origin; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure
D015342:DNA Probes; D004279:DNA, Viral; D009698:Nucleoproteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate