The iron–sulphur cluster in human DNA2 is required for all biochemical activities of DNA2

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.

Mitochondrial iron–sulfur clusters: Structure, function, and an emerging role in vascular biology

“…These clusters also have regulatory roles, modulating gene expression in response to oxidative stress (via superoxide response (SoxR) proteins), oxygen levels (via fumarate-nitrate reduction (FNR) proteins), as well as iron levels (via Iron Response Proteins including IRP1 and IRP2) [ [9] , [10] , [11] ]. Fe–S clusters have also been observed to play important roles in DNA metabolism, coordinating protein conformational changes during DNA replication and repair, as found in primases, helicases, nucleases, and polymerases [ [12] , [13] , [14] , [15] , [16] ]. Within these enzymes, these clusters are involved in maintaining structural stability, and are also known to play roles in DNA binding, unwinding, and exonuclease activity.…”

The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

“…One explanation for the reduction in heavy chain protein in ABCB7-deficient pro-B cells may be inability to repair double-stranded DNA breaks during VDJ recombination. Importantly, the Fe-S-GSH intermediates transported by ABCB7 mature into cofactors used in numerous DNA replication and damage repair enzymes including DNA primase, all replicative DNA polymerases, Dna2, FancJ, XPD, Endo III, and MutY ( Baranovskiy et al, 2018 ; Cunningham et al, 1989 ; Fuss et al, 2015 ; Klinge et al, 2007 ; Mariotti et al, 2020 ; Netz et al, 2011 ; Porello et al, 1998 ; Puig et al, 2017 ; Rudolf et al, 2006 ). As recombination only occurs in nonproliferating cells due to Rag2 protein degradation ( Lin and Desiderio, 1994 ), DNA damage was assessed by analyzing pH2A.X (γH2A.X) expression ( Smith et al, 2010 ) in parallel with a 3 hr EdU pulse to identify proliferating pro-B cells.…”

“…The Fe-S-GSH intermediates transported by ABCB7 mature in the cytoplasm where they are then used as critical cofactors for numerous enzymes involved in DNA replication and damage repair, including DNA primase, all replicative DNA polymerases, the helicases Dna2, FancJ, and XPD, and the glycosylases Endo III and MutY ( Baranovskiy et al, 2018 ; Cunningham et al, 1989 ; Fuss et al, 2015 ; Klinge et al, 2007 ; Mariotti et al, 2020 ; Netz et al, 2011 ; Porello et al, 1998 ; Puig et al, 2017 ; Rudolf et al, 2006 ). Because we observed pH2A.X was highly expressed in EdU + cells, we hypothesize that DNA damage is being induced during replication due to defects caused by aberrant Fe-S cluster transport in the absence of ABCB7.…”

“…Fe-S clusters are important cofactors for numerous proteins involved in cellular metabolism, DNA replication, and DNA damage repair ( Fuss et al, 2015 ; Lane et al, 2015 ; Lill and Mühlenhoff, 2006 ). These proteins include ferredoxin ( Lange et al, 2000 ); components of NADH:ubiquinone oxidoreductase (Complex I) ( Ohnishi, 1998 ); DNA primase ( Klinge et al, 2007 ); all of the replicative DNA polymerases ( Netz et al, 2011 , reviewed by Baranovskiy et al, 2018 ; Puig et al, 2017 ); the helicases Dna2, FancJ, and XPD ( Mariotti et al, 2020 ; Rudolf et al, 2006 ); and the glycosylases Endo III and MutY ( Cunningham et al, 1989 ; Porello et al, 1998 ). Fe-S cluster intermediates are first biosynthesized in the mitochondria, after which they are transported to the cytoplasm for maturation ( Maio and Rouault, 2015 ; Rouault, 2012 ).…”

Sulfur Administration in Fe–S Cluster Homeostasis

“…The Fe–S clusters are cofactors of proteins involved in maintaining genome integrity [ 35 ]. DNA-binding proteins have the Fe–S clusters in their structure, and thus their activity is granted [ 19 , 36 ] ( Figure 2 ). They are involved in tRNA modification [ 7 , 37 ].…”