The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA.

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.