Adeno-associated virus Rep78/Rep68 promotes localized melting of the rep binding element in the absence of adenosine triphosphate.
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.
Lou
Hua Jane
HJ
Department of Chemistry and McKnight Brain Institute, Shands Cancer Center, University of Florida, Gainesville, FL 32611, USA.
Brister
J Rodney
JR
Li
Jianwei Jeffery
JJ
Chen
Weijun
W
Muzyczka
Nicholas
N
Tan
Weihong
W
D000255:Adenosine Triphosphate; D000256:Adenoviridae / Q000737:chemistry / Q000235:genetics; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D044367:Phase Transition; D012045:Regulatory Sequences, Nucleic Acid; D014764:Viral Proteins / Q000378:metabolism / Q000502:physiology*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D000255:Adenosine Triphosphate; D004265:DNA Helicases