Structure, function and evolution of the mitochondrial replicative DNA helicase (570.1)

Abstract

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N‐terminal domain (NTD) and C‐terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein‐protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N‐terminal truncated variants: a full‐length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron‐sulfur cluster, most likely a 2Fe‐2S, as determined by mass spectrometry, and UV‐visible and atomic absorption spectroscopy. Mutagenesis studies show that the four conserved cysteine residues in the ZBD responsible for binding zinc in T7gp4, bind the iron‐sulfur cluster. We extend our biochemical studies in vivo through several approaches to identify partner proteins of the Drosophila mtDNA helicase that may elucidate the functional role of its iron‐sulfur cluster.Grant Funding Source: NIH GM45295