DNA and RNA Binding by the Mitochondrial Lon Protease Is Regulated by Nucleotide and Protein Substrate

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (14), p.13902-13910
• Main Authors: Liu, Tong, Lu, Bin, Lee, Irene, Ondrovicová, Gabriela, Kutejová, Eva, Suzuki, Carolyn K
• Format: Article
• Language: English
• Subjects: ATP-Dependent Proteases; Cations - metabolism; DNA Helicases; DNA Polymerase gamma; DNA Primase - metabolism; DNA, Mitochondrial - metabolism; DNA-Binding Proteins - metabolism; DNA-Directed DNA Polymerase - metabolism; Guanine - metabolism; Heat-Shock Proteins - metabolism; Humans; In Vitro Techniques; Mitochondria - enzymology; Mitochondria - genetics; Mitochondrial Proteins; Recombinant Proteins - metabolism; RNA-Binding Proteins - metabolism; Serine Endopeptidases - metabolism; Substrate Specificity; Uracil - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309642200

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro , human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo . Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase γ and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.