title: Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding.
abstract: Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Lo
authors.forename: Shih-Wei
authors.initials: SW
authors.lastname: Liu
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Li
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Lizz
authors.forename: Chwan-Deng
authors.initials: CD
authors.lastname: Hsiao
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics*; D019915:DNA Primase / Q000378:metabolism; D056506:Geobacillus / Q000201:enzymology*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D050505:Mutant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D019915:DNA Primase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases