Abstract
The EBNA1 protein of Epstein-Barr virus (EBV) binds to and activates DNA replication from the EBV latent origin of replication, oriP, via a direct interaction with the two noncontiguous subelements of oriP. The EBNA1 molecules bound to the oriP subelements interact efficiently with each other by a DNA looping mechanism. We have previously mapped a region of EBNA1 (termed the looping region) that is required to mediate the interaction of the EBNA1 molecules bound to the oriP subelements. We now demonstrate that two fragments of this region of EBNA1, which consist largely of an eight amino acid repeat, can mediate homotypic interactions when transferred to another DNA-binding protein. Protein interactions mediated by the EBNA1 looping region appear to be dependent on DNA binding since these interactions were detected between DNA-bound forms of the proteins only.
Highlights
The interaction at a distance between DNA-bound proteins by a DNA looping mechanism has been reported to underlie a variety of biological processes including transcription, DNA replication, and site-specific DNA recombination (reviewed in Matthews (1992) and Schleif (1992))
EBNA1 Fragments Can Direct the Interaction between DNAbound GAL4 Proteins—We have demonstrated previously that the interaction at a distance of DNA-bound EBNA1 molecules requires part of the internal basic region of EBNA1 between amino acids 327 and 377 (see Fig. 1; Frappier et al, 1994 (note that the aminoterminal amino acid numbers reported in Frappier et al (1994) are off by 1, i.e. the 350 – 641 truncation mutant is really 351– 641)).In order to determine whether EBNA1 amino acids 351–377 are sufficient for this interaction, we fused this portion of EBNA1 to the GAL4 DNA binding domain (Fig. 1)
The fusion proteins were overproduced in E. coli, purified to Ͼ75% purity, and assayed for DNA binding activity by electrophoretic mobility shift using DNA fragments containing 5 tandem GAL4 binding sites.The amount of each protein required to bind 50% of the DNA molecules was tested for the ability to mediate interactions between the GAL4 binding sites in a ligation enhancement assay (Fig. 2)
Summary
The interaction at a distance between DNA-bound proteins by a DNA looping mechanism has been reported to underlie a variety of biological processes including transcription, DNA replication, and site-specific DNA recombination (reviewed in Matthews (1992) and Schleif (1992)). The FR contains 20 EBNA1 binding sites and, when bound by EBNA1, activates DNA replication from the DS element, enhances transcription from viral promoters, and governs the stable segregation of the EBV episomes during cell division (Reisman et al, 1985; Reisman and Sugden, 1986; Krysan et al, 1989; Gahn and Sugden, 1995). In order to better understand the mechanism by which these interactions occur, we have used mutational analyses to identify a region of EBNA1 (termed the DNA looping region) that is required to mediate the interaction between FR- and DS-bound EBNA1 molecules (Goldsmith et al, 1993; Frappier et al, 1994) This region is distinct from the DNA binding and dimerization domains of EBNA1 and likely directs the homotypic protein interaction. We demonstrate that the DNA looping region is composed of at least two independent domains that are sufficient to mediate homotypic protein interactions and that retain the ability to mediate these interactions when transferred to another DNA-binding protein
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
