The roles of the residues on the channel beta-hairpin and loop structures of simian virus 40 hexameric helicase.
Simian virus 40 large tumor antigen is required for DNA unwinding during viral replication. The helicase-active form of large tumor antigen is a ring-shaped hexamer/double hexamer, which has a positively charged hexameric channel for interacting with DNA. On the hexameric channel surface are six beta-hairpin structures and loops, emanating from each of the six subunits. At the tips of the beta-hairpin and the loop structures are two ring-shaped residues, H513 and F459, respectively. Additionally, two positively charged residues, K512 and K516, are near the tip of the beta-hairpin. The positions of these ring-shaped and positively charged residues suggest that they may play a role in binding DNA for helicase function. To understand the roles of these residues in helicase function, we obtained a set of mutants and examined various activities, including oligomerization, ATPase, DNA binding, and helicase activities. We found that substitution of these residues by Ala abolished helicase activity. Extensive mutagenesis showed that substitutions by ring-shaped residues (W and Y) at position F459 and by residues with hydrophobic or long aliphatic side chains (W, Y, F, L, M, and R) at position H513 supported helicase activity. Our study demonstrated that the four residues (F459, H513, K512, and K516) play a critical role in interacting with DNA for helicase function. The results suggest a possible mechanism to explain how these residues, as well as the beta-hairpin and the loop structures on which the residues reside, participate in binding and translocating DNA for origin melting and unwinding.
Shen
Jingping
J
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089-1340, USA.
Gai
Dahai
D
Patrick
Aaron
A
Greenleaf
William B
WB
Chen
Xiaojiang S
XS
D000957:Antigens, Viral, Tumor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D003460:Crystallization; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008958:Models, Molecular; D016296:Mutagenesis; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D017434:Protein Structure, Tertiary; D013539:Simian virus 40 / Q000235:genetics*
D000957:Antigens, Viral, Tumor; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D004247:DNA; D004265:DNA Helicases