title: The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA.
abstract: The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48824 and the Department of Biology and Biochemistry, Birzeit University, P. O. Box 14, West Bank 627, Palestine.
authors.forename: Johnny
authors.initials: J
authors.lastname: Stiban
authors.affiliation: From the Department of Biochemistry and Molecular Biology, and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Farnum
authors.affiliation: From the Department of Biochemistry and Molecular Biology, and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Stacy L
authors.initials: SL
authors.lastname: Hovde
authors.affiliation: From the Department of Biochemistry and Molecular Biology, and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan 48824 and lskaguni@msu.edu.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D004272:DNA, Mitochondrial; D007506:Iron-Sulfur Proteins; D004265:DNA Helicases
keywords: DNA Helicase; DNA Replication; DNA-binding Protein; Drosophila; Iron-Sulfur Protein; Mitochondria; Mitochondrial DNA (mtDNA)