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

Objectives and Methods: Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) , which is essential for EBV latency, homodimerizes and binds to the EBV re plication 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 great ly 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 h elix-deforming substitution in a-helix III (568-582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and cons erved amino acids were found between alpha -helix II and recognition helice s of papillomavirus E2 proteins. Conclusions: The basic DNA contact region is crucial for the specific interaction of EBNA-1 with a single binding sit e. alpha -Helix I-477 is indispensable for oriP binding, and alpha -helix I II568 contributes to the homodimeric structure of EBNA-1. alpha -Helix II51 4 contributes to oriP binding, perhaps changing its alignment with DNA. Cop yright (C) 2001 S. Karger AG, Basel.