Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2

The Journal of Biological Chemistry
Miran Yoon-RobartsR Michael Linden

Abstract

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...Continue Reading

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Citations

Nov 16, 2012·Journal of Virology·Francisco Zarate-PerezCarlos R Escalante
Jul 14, 2009·PLoS Pathogens·Jorge Mansilla-SotoR Michael Linden
Jan 8, 2011·Future Virology·Els Henckaerts, R Michael Linden
Aug 21, 2015·Journal of Virology·Michael SeyffertCornel Fraefel
Sep 19, 2019·Human Gene Therapy·Suriyasri SubramanianSusan L Hafenstein
Apr 19, 2020·Human Gene Therapy·Anna C Maurer, Matthew D Weitzman
Jun 15, 2017·Bio-protocol·Francisco Zarate-PerezCarlos R Escalante
Jul 22, 2021·Journal of Virology·Mario MietzschMavis Agbandje-McKenna
Jan 11, 2007·Biochemistry·Susan S DignamJohn David Dignam

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