Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010

Unwinding of double-stranded DNA into single-stranded intermediates require d for various fundamental life processes is catalyzed by helicases, a famil y of mono-, di- or hexameric motor proteins fueled by nucleoside triphospha te hydrolysis. The three-dimensional crystal structure of the hexameric hel icase RepA encoded by plasmid RSF1010 has been determined by X-ray diffract ion at 2.4 Angstrom resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a similar to 17 Angstrom wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homo logs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and th e helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex , ATP molecules are located at the subunit interfaces and clearly define ad enine-binding and ATPase catalytic sites formed by amino acid residues loca ted on adjacent monomers; most remarkable is the "arginine finger" Arg207 c ontributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrol ysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published as ymmetric structure of the bacteriophage T7 gp4 helicase domain. (C) 2001 Ac ademic Press.