Multiple regions within EBNA1 can link DNAs.
Epstein-Barr virus nuclear antigen 1 (EBNA1) can bind specifically to two clusters of sites within the Epstein-Barr virus plasmid origin of DNA replication (oriP). EBNA1 activates DNA replication mediated by oriP and can also activate transcription and retain DNA in cells when bound site specifically. EBNA1 bound to oriP physically links the two clusters of EBNA1-binding sites, resulting in loop formation by the intervening DNA. To elucidate the contribution of DNA linking by EBNA1 to its biological activities, we identified regions within it that can independently link DNAs to which they are bound. An electrophoretic mobility shift assay was used to detect this activity. Proteins which link DNA aggregate that DNA into large lattices. Proteins which cannot link DNA but still bind to DNA retard the mobility of that DNA but do not cause it to form lattices. Amino-terminal truncations were used to map the amino-terminal limit of a minimal DNA-linking domain approximately to amino acid 372 of EBNA1. To map the carboxy-terminal limit of this minimal domain, fusion proteins containing the DNA-binding domain of GAL4 and fragments of EBNA1 were generated and studied. This approach identified the carboxy-terminal limit of this minimal domain to be approximately amino acid 391 and verified its amino-terminal limit. Internal deletions within a truncated EBNA1 derivative verified the importance of this region. Two additional fragments of EBNA1, each of which independently conferred DNA-linking activity on the domain of GAL4 which binds DNA, were identified within amino acids 54 to 89 and amino acids 331 to 361. Therefore, EBNA1 contains at least three regions that can act independently to link DNAs and that may act in concert within intact EBNA1.
Mackey
D
D
McArdle Laboratory for Cancer Research, Department of Oncology, Medical School, University of Wisconsin-Madison, USA.
Middleton
T
T
Sugden
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018741:Replication Origin; D015183:Restriction Mapping; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection
D000956:Antigens, Viral; D015342:DNA Probes; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA