STABILIZATION OF THE EBNA1-PROTEIN ON THE EPSTEIN-BARR-VIRUS LATENT ORIGIN OF DNA-REPLICATION BY A DNA LOOPING MECHANISM

DNA replication from the Epstein-Barr virus latent origin of replicati on, oriP, is activated by Epstein-Barr nuclear antigen 1 (EBNA1). This activation involves the binding of EBNA1 dimers to multiple sites pre sent in the two noncontiguous functional elements of oriP, the dyad sy mmetry element (DS) from which replication initiates, and the family o f repeats (FR) enhancer element. EBNA1 complexes formed on the FR and DS elements of oriP interact by a DNA looping mechanism. This interact ion requires EBNA1 sequences in addition to those required for DNA bin ding and dimerization. To map the EBNA1 sequences required for the eff icient interaction of FR- and DS-bound EBNA1 complexes, we have overpr oduced in Escherichia coli and purified a series of EBNA1 N-terminal t runcation mutants, all of which retain the DNA binding and dimerizatio n domains. The results of electron microscopy and ligation-enhancement assays using these mutants indicated that EBNA1 sequences between ami no acids 350 and 361 are required for the efficient interaction of FR- and DS-bound EBNA1 complexes. EBNA1-mediated FR-DS interactions were shown to stabilize EBNA1 binding to the DS element, while EBNA1-mediat ed DS-DS interactions did not. These results suggest that the stabiliz ation of EBNA1 on the DS element, which occurs as a result of EBNA1-me diated oriP looping, may be important for the activation of DNA replic ation from the DS element.