The iron\u2013sulphur cluster in human DNA2 is required for all biochemical activities of DNA2

Laura Mariotti,Sandra Kummer,Richard E Lutz,Sebastian Wild,Ilaria Ceppi,Alessandra Piceni,Kerstin Gari,Giulia Brunoldi,Petr Cejka

doi:10.1038/s42003-020-1048-4

Laura Mariotti, Sandra Kummer + Show 7 more

Open Access

https://doi.org/10.1038/s42003-020-1048-4

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Abstract

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2’s nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.

Highlights

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks
We demonstrate that loss of the FeS cluster in human DNA2 induces a conformational change that likely distorts the DNA-binding tunnel and impairs DNA binding
To obtain FeS cluster-deficient versions of human DNA2, we replaced each of the four ligating cysteines with serine residues (Fig. 1a)

Summary

Introduction

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. In some DNA maintenance proteins, FeS clusters act as important structural components involved in protein complex formation[12] or supporting catalytic and non-catalytic activities of their host proteins[7,8,13] In others, they seem to have an active redox role, such as in the bacterial DNA damage-inducible protein DinG, where reduction of its FeS cluster reversibly inactivates its helicase activity[14]. We discover that DNA2 is redoxregulated, but, in contrast to the helicase DinG14, this redox sensitivity is not mediated by its FeS cluster This surprising finding adds DNA2 to the growing list of nuclear proteins which may act as redox sensors

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