Molecular Interplay between the Replicative Helicase DnaC and Its Loader Protein DnaI from Geobacillus kaustophilus

• Published in: Journal of molecular biology 2009-11, Vol.393 (5), p.1056-1069
• Main Authors: Tsai, Kuang-Lei, Lo, Yu-Hua, Sun, Yuh-Ju, Hsiao, Chwan-Deng
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Amino Acid Sequence; Bacillus - enzymology; Bacillus subtilis; Bacterial Proteins - chemistry; Binding Sites; Chromatography, Gel; Crystallography, X-Ray; DNA Helicases - chemistry; DNA Replication - drug effects; DNA, Single-Stranded - metabolism; DnaC; DnaI; Electrophoresis, Polyacrylamide Gel; Geobacillus; helicase; loader; Models, Molecular; Molecular Sequence Data; Nucleotides - pharmacology; Protein Binding - drug effects; Protein Structure, Secondary; Protein Structure, Tertiary; replication; Sequence Alignment; Surface Plasmon Resonance
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2009.09.002

Helicase loading factors are thought to transfer the hexameric ring-shaped helicases onto the replication fork during DNA replication. However, the mechanism of helicase transfer onto DNA remains unclear. In Bacillus subtilis, the protein DnaI, which belongs to the AAA+ family of ATPases, is responsible for delivering the hexameric helicase DnaC onto DNA. Here we investigated the interaction between DnaC and DnaI from Geobacillus kaustophilus HTA426 ( GkDnaC and GkDnaI, respectively) and determined that GkDnaI forms a stable complex with GkDnaC with an apparent stoichiometry of GkDnaC 6– GkDnaI 6 in the absence of ATP. Surface plasmon resonance analysis indicated that GkDnaI facilitates loading of GkDnaC onto single-stranded DNA (ssDNA) and supports complex formation with ssDNA in the presence of ATP. Additionally, the GkDnaI C-terminal AAA+ domain alone could bind ssDNA, and binding was modulated by nucleotides. We also determined the crystal structure of the C-terminal AAA+ domain of GkDnaI in complex with ADP at 2.5 Å resolution. The structure not only delineates the binding of ADP in the expected Walker A and B motifs but also reveals a positively charged region that may be involved in ssDNA binding. These findings provide insight into the mechanism of replicative helicase loading onto ssDNA.