Abstract
Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin of DNA replication, oriP. EBNA1 binds cooperatively to four recognition sites in the dyad symmetry (DS) element of oriP, causing alterations in the origin DNA structure, which can be detected by the increased sensitivity of one Thy residue in two of the binding sites to permanganate oxidation. To better understand the significance of this EBNA1-induced origin distortion, we have investigated the DNA sequence and EBNA1 amino acid requirements for permanganate sensitivity. We have shown that the EBNA1 DNA binding and dimerization domains are sufficient to induce permanganate sensitivity and that amino acids 463-467, which form an extended chain that travels along the minor groove of the EBNA1 recognition site, play an important role in generating the DNA distortion. The EBNA1-induced permanganate sensitivity is independent of cooperative interactions between EBNA1 molecules on the origin and requires a specific sequence within the EBNA1 binding site. Using synthetic EBNA1 binding sites, we found that the inversion of a single AT base pair in the EBNA1 recognition sequence is sufficient to confer EBNA1-induced permanganate sensitivity. These studies indicate that permanganate oxidation can detect very minor alterations in DNA structure.
Highlights
The first step in the initiation of DNA synthesis in all replication systems involves the melting of the origin DNA
To better understand how localized DNA distortions caused by origin-binding proteins contribute to origin melting, we have examined the structural changes in origin DNA induced by Epstein-Barr virus nuclear antigen 1 (EBNA1)
EBNA1 Amino Acids Required for dyad symmetry (DS) DNA Distortion— Structural changes in the DS element of oriP, which can be detected by permanganate oxidation, are induced by wild type and internally deleted versions of EBNA1 [15, 18, 19]
Summary
DNA—The construction of pGEMdyad, which contains the DS element of oriP, and pGEMs1, which contains only EBNA1 binding site 1 from the DS element, have been previously described [17, 21]. Plasmids containing the palindromic EBNA1 consensus binding site (CS) or the consensus binding site with an inversion of the AT base pairs at position 6 and Ϫ6 (CSA/T) were constructed from the oligonucleotides 5Ј-GGGTAGCATATGCTACCC-3Ј and 5Ј-GGGAAGCATATGCTTCCC-3Ј, reconsensus binding site; CSA/T, consensus binding site with an inversion of the AT base pairs; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; DMS, dimethyl sulfate. Extension products were separated on an 8% polyacrylamide/50% urea sequencing gel and visualized by autoradiography
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