Amino acid substitution analyses of the DNA contact region, two amphipathic alpha-helices and a recognition-helix-like helix outside the dimeric beta-barrel of Epstein-Barr virus nuclear antigen 1.
OBJECTIVE
Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), which is essential for EBV latency, homodimerizes and binds to the EBV replication origin, oriP. We analyzed the dimerization/DNA-binding domain of EBNA-1 by random and site-directed amino acid substitution.


RESULTS
Random point mutations that resulted in reduced DNA binding clustered in the DNA contact region (a.a. 461-473) and at or near the termini of alpha-helix II (514-527). Three substitutions of Gly in the DNA contact region each greatly reduced binding to a single binding site oligonucleotide. Substitutions at and near the termini of alpha-helix II diminished DNA binding. A helix-deforming substitution in alpha-helix I (477-489) blocked DNA binding. A helix-deforming substitution in alpha-helix III (568-582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and conserved amino acids were found between alpha-helix II and recognition helices of papillomavirus E2 proteins.


CONCLUSIONS
The basic DNA contact region is crucial for the specific interaction of EBNA-1 with a single binding site. Alpha-helix I477 is indispensable for oriP binding, and alpha-helix III568 contributes to the homodimeric structure of EBNA-1. Alpha-helix II514 contributes to oriP binding, perhaps changing its alignment with DNA.
Fujita
T
T
Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
Ikeda
M
M
Kusano
S
S
Yamazaki
M
M
Ito
S
S
Obayashi
M
M
Yanagi
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D011993:Recombinant Fusion Proteins; C417029:EBV-encoded nuclear antigen 1; D005998:Glycine