Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase.

Minyoung So,Stacy L Hovde,Grzegorz L Ciesielski,Laurie S Kaguni,Johnny Stiban

doi:10.3389/fgene.2021.790521

Abstract

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Highlights

Iron-sulfur clusters (ISCs) are ancient cofactors thought to be involved in the emergence of the origin of life (Koonin and Martin, 2005)
We evaluate the possibility that it may serve as the ISC transfer protein for Dm mtDNA helicase by documenting its mitochondrial localization, cofactorindependent dimerization and membrane binding properties
We demonstrated previously the presence of an ISC in Dm mtDNA helicase (Stiban et al, 2014), though the possibility remained that cluster insertion might be an artefact of the E. coli overexpression system

Summary

Introduction

Iron-sulfur clusters (ISCs) are ancient cofactors thought to be involved in the emergence of the origin of life (Koonin and Martin, 2005). ISCs have been identified in diverse nucleic acid processing enzymes such as primases (Weiner et al, 2007; O’Brien et al, 2017), polymerases (Netz et al, 2012), helicases (Rudolf et al, 2006; Fan et al, 2008; Stiban et al, 2014), nucleases (Yeeles et al, 2009; Pokharel and Campbell, 2012; Sparks et al, 2012), glycosylases (Boal et al, 2009), tRNA thiolating enzymes (Romsang et al, 2018), and transcription factors (Khoroshilova et al, 1997). Charge transfer between [4Fe-4S] proteins and DNA was shown to be unidirectional and kinetically unfavorable in the opposite direction, such that oxidants or reductants may be necessary (Teo et al, 2019)

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Conclusion