Mechanism of DNA Recognition at a Viral Replication Origin

Abstract

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1 , to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol ؊1 , with 11.9 kcal mol ؊1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1 452-641 ⅐DNA complex is accompanied by a heat capacity change of ؊1.22 kcal mol ؊1 K ؊1 , a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1⅐DNA complex conformers are in slow equilibrium; one dissociates slowly (t1 ⁄ 2 ϳ 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1 ⁄ 2 ϳ 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6 ؋ 10 8 M ؊1 s ؊1 , which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of ϳ2.0 s ؊1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.Initiation of DNA replication from cellular and viral origins rely on origin binding proteins (OBPs), 2 also referred to as initiator proteins. These OBPs nucleate the replication machinery by recruiting various proteins and induce the local distortion of the DNA as a previous step for unwinding. There are two groups of viral OBPs, one of them is represented by the EpsteinBarr nuclear antigen 1 (EBNA1) and by the E2 protein from papillomaviruses, both of which bind to the origin but rely on other factors to melt the DNA (1). The second group consists of viral OBPs that have both DNA binding activity and helicase activity, represented by the SV-40 T antigen and the E1 helicase from papillomavirus. The second group of OBPs facilitates the binding and cooperates with those of the first group, such as the case of papillomavirus E2 that cooperates with the E1 helicase (1).EBNA1 is the only protein expressed in all types of EBV latent infection (2). It binds to the EBV replication origin oriP where it plays several roles: initiation of DNA replication, segregation of EBV episomes, and transactivation of latent viral gene expression (3-5). All these functions require the binding of the EBNA1 DNA binding domain to specific 18-bp DNA rec...

Experimental snapshots of a protein-DNA binding landscape

“…Discussion Specific protein-DNA-binding at equilibrium is often a deceptively simple, two-state process in which only the unbound reagents and the final complex are populated (4)(5)(6)(7)(8). However, the few kinetic mechanisms determined to date revealed a richer picture, with parallel routes and transient intermediates (4)(5)(6)(7)(8).…”

“…However, the few kinetic mechanisms determined to date revealed a richer picture, with parallel routes and transient intermediates (4)(5)(6)(7)(8). In the absence of high-resolution kinetic work, structural studies suggested that multistate routes for protein-DNA-binding start with the formation of nonspecific interactions with the DNA backbone (1,(9)(10)(11)(12)(13)(14)(15)(16)(17), and that some dual-role residues may form transient nonnative interactions along the route (13-15, 17).…”

“…It is, thus, generally accepted that these proteins first bind nonspecifically to genomic DNA which speeds up the search for their target sequence through facilitated diffusion (3). The kinetics of DNA sequence recognition (3,4) often involve multistate kinetic routes with populated intermediates (4)(5)(6)(7)(8). The build up of molecular interactions along multistate routes has been deduced indirectly from structural and thermodynamic studies (1)(2)(3)(9)(10)(11)(12)(13)(14)(15)(16)(17), but kinetic characterization of intermediates and transition state ensembles is still scarce.…”

Transition state for protein–DNA recognition

“…Some proteins diffuse mainly in 1D over long distances (2), whereas others favor a 3D pathway (3). Once at the target site, proteins without an enzymatic activity may bind to DNA along a single kinetic route (4)(5)(6)(7)(8)(9) or parallel routes (10,11). Each route may either be two-state (4-6, 10) or include populated kinetic intermediates (7)(8)(9)(10)(11).…”

“…Once at the target site, proteins without an enzymatic activity may bind to DNA along a single kinetic route (4)(5)(6)(7)(8)(9) or parallel routes (10,11). Each route may either be two-state (4-6, 10) or include populated kinetic intermediates (7)(8)(9)(10)(11). In the final consolidated complex, sequence recognition is mediated by specific contacts between protein residues and DNA bases (direct sequence readout) and by the conformational energetics of noncontacted bases (indirect sequence readout) (12)(13)(14)(15)(16).…”