Adeno‐Associated Virus Rep78/Rep68 Promotes Localized Melting of the Rep Binding Element in the Absence of Adenosine Triphosphate

Abstract

We have applied fluorescence anisotropy and molecular beacon fluorescence methods to study the interactions between the Adeno-associated virus Rep78/Rep68 protein and the 23-bp Rep binding element (RBE). Rep78/Rep68 stably interacted with both the single- and double-stranded conformations of the RBE, but the interaction mechanisms of single- and double-stranded DNA appeared to be fundamentally different. The stoichiometry of Rep78 association with both the separate top and bottom strands of the RBE was 1:1, and the relative dissociation constant (K(D)) values of these associations were calculated to be 2.3x10(-8) and 3.2x10(-8) M, respectively. In contrast, the stoichiometry of Rep78 association with the double-stranded RBE was 2:1, and the dissociation constant was determined to be 4.2x10(-15) M(2). Moreover, Rep78/Rep68 interaction with the 23-bp duplex RBE appeared to cause localized melting of the double-stranded DNA substrate in the absence of adenosine triphosphate (ATP). This melting activity showed slower kinetics than binding and may contribute to the initiation of ATP-dependent Rep78 helicase activity.

A pH-insensitive, ratiometric chemosensor for citrate using europium luminescence

“…The 1 : 1 stoichiometry of complexation was confirmed by a Job plot, and an apparent binding constant of 3.3 (¡0.6) 6 10 6 M 21 was estimated by analysing the binding isotherm. { 15 This high affinity was consistent with the observation by electrospray mass spectrometry of the 1 : 1 citrate-bound species; for example at m/z 1743 ([EuL 1 ](CF 3 SO 3 ) 3 + citrate + 4Na) + , 1571 ([EuL 1 ](CF 3 SO 3 ) 2 + citrate + 3Na) + and 1229 ([EuL 1 citrate] + Na) + . The luminescence intensity change between the DJ 5 2 and DJ 5 0 transitions as a function of citrate concentration permitted a ratiometric measurement.…”

Parvoviruses: Small Does Not Mean Simple

“…This extended-form duplex is then melted and refolded into two hairpins (step iv), creating a rabbit-ear structure that pairs the 3 nucleotide of the newly synthesized DNA with an internal base, effectively reversing the path of the fork and redirecting it back along the internal coding sequences (step v). Both unfolding and refolding the hairpins require NS1, which binds site specifically to duplex motifs in the telomere and requires a functional SF3 helicase domain to assist in melting the duplex (100,101). The fork then progresses back along the monomeric duplex, displacing the original negative strand and replacing it with a covalently continuous new strand.…”

Parvovirus Diversity and DNA Damage Responses

“…To support DNA replication, all telomeres must encode multipartite replication origins and engage in hinge-like hairpin rearrangements, which require local melting induced by site-specific binding of the initiator proteins (Willwand et al 2002;Lou et al 2004). In addition, axial asymmetries in the hairpins, most commonly manifest as T-or Y-shaped structures, likely facilitating hairpin unfolding and refolding by lowering the free energy of the duplex.…”