Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication.
The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus is a multifunctional protein with important roles in both transcriptional regulation and episomal maintenance. LANA is also a DNA-binding protein and has been shown to specifically bind to a region within the terminal repeat. Here, we have performed a detailed analysis of the DNA-binding activity of LANA and show that it binds two sites separated by 22 bp. We used electrophoretic mobility shift assay to quantitatively analyze the binding sites and determined that the K(d) of the high affinity site is 1.51 +/- 0.16 nm. Examination of the contribution of nucleotides near the ends of the site showed that the core binding site consists of 16 bp, 13 of which are conserved between both sites. Analysis of the affinity of each site alone and in tandem revealed that the binding to the second site is primarily due to cooperativity with the first site. Using deletion and point mutations, we show that both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication. In addition, we show that the ability of LANA to carry out these functions is directly proportional to its affinity for the sites in this region. The affinities, spacing, and cooperative binding between the two sites is similar to that of the Epstein-Barr virus dyad symmetry element oriP, suggesting a requirement for such an element in latent replication of these related DNA tumor viruses.
Garber
Alexander C
AC
Division of Hematology/Oncology, Department of Molecular Biology and Microbiology, Case Western Reserve University, 2109 Adelbert Road, Cleveland, OH 44106, USA.
Hu
Jianhong
J
Renne
Rolf
R
D000956:Antigens, Viral; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D014162:Transfection
D000956:Antigens, Viral; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen; D004247:DNA