Amino acid substitution analyses of the DNA contact region, two amphipathic α-helices and a recognition-helix-like helix outside the dimeric β-barrel of Epstein-Barr virus nuclear antigen 1

Objectives and Methods: 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 α-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 α-helix II diminished DNA binding. A helix-deforming substitution in α-helix I (477-489) blocked DNA binding. A helix-deforming substitution in α-helix III (568-582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and conserved amino acids were found between α-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. α-Helix I₄₇₇ is indispensable for oriP binding, and α-helix III₅₆₈ contributes to the homodimeric structure of EBNA-1. α-Helix II₅₁₄ contributes to oriP binding, perhaps changing its alignment with DNA.