Molecular interplay between the replicative helicase DnaC and its loader protein DnaI from Geobacillus kaustophilus.
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 A 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.
Tsai
Kuang-Lei
KL
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, ROC.
Lo
Yu-Hua
YH
Sun
Yuh-Ju
YJ
Hsiao
Chwan-Deng
CD
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry*; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009711:Nucleotides; D000251:Adenosine Triphosphatases; D004265:DNA Helicases