The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities.
The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg(2+) or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.
Farge
Géraldine
G
Division of Metabolic Diseases, Karolinska Institutet, Novum, SE-141 86 Stockholm, Sweden.
Holmlund
Teresa
T
Khvorostova
Julia
J
Rofougaran
Reza
R
Hofer
Anders
A
Falkenberg
Maria
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000074002:DNA Polymerase gamma; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D024101:Mitochondrial Proteins; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
D004277:DNA, Single-Stranded; D024101:Mitochondrial Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; C431576:TWNK protein, human