Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2.
We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.
Yoon-Robarts
Miran
M
Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Blouin
Amanda G
AG
Bleker
Svenja
S
Kleinschmidt
Jürgen A
JA
Aggarwal
Aneel K
AK
Escalante
Carlos R
CR
Linden
R Michael
RM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary
D004247:DNA; D004265:DNA Helicases; C477740:Rep40 protein, adeno-associated virus; D008239:Lysine