The DNA Repair Helicases XPD and FancJ Have Essential Iron-Sulfur Domains

Abstract

DNA helicases are essential components of the cellular machinery for DNA replication, recombination, repair, and transcription. The XPD and FancJ proteins are related helicases involved in the nucleotide excision repair (NER) and Fanconi anemia repair pathways, respectively. We demonstrate that both proteins have a conserved domain near the N terminus that includes an iron-sulfur (Fe-S) cluster. Three absolutely conserved cysteine residues provide ligands for the Fe-S cluster, which is essential for the helicase activity of XPD. Yeast strains harboring mutations in the Fe-S domain of Rad3 (yeast XPD) are defective in excision repair of UV photoproducts. Clinically relevant mutations in patients with trichothiodystrophy (TTD) and Fanconi anemia disrupt the Fe-S clusters of XPD and FancJ and thereby abolish helicase activity.

Analysis of iron–sulfur protein maturation in eukaryotes

“…The functional significance of the ISC and CIA machineries has gained progressive interest since a number of essential cytosolic and nuclear Fe/S proteins have been identified, the function of which may critically depend on their Fe/S cofactors [29][30][31] . In fact, the presence of essential Fe/S clusters in these proteins may explain the indispensable character of mitochondria and the ISC and CIA machineries for cell viability 29 .…”

“…Ribosome maturation and protein translation require the 2Â [4Fe-4S] cluster-containing protein Rli1, which resides in both cytosol and nucleus 29,32 . Other essential Fe/S proteins are involved in the integrity, maintenance and repair of DNA, but the presence of Fe/S clusters in these proteins in vivo has not been verified yet 30,31 . Replication of the lagging strand critically depends on the primase complex, which includes the Fe/S cluster-containing large subunit Pri2 (ref.…”

“…The DNA helicase Rad3 (XPD in humans) is one of the ten subunits of the RNA polymerase transcription factor IIH and is vital for nucleotide excision DNA repair 33 . A prokaryotic homolog of Rad3 has been shown by biochemical and structural studies to contain an essential [4Fe-4S] cluster inserted into its helicase fold 31,34 . This protocol describes various experimental approaches to verify that these proteins in fact contain Fe/S clusters in the living cell.…”

“…Currently, the majority of novel Fe/S proteins are identified by isolation after heterologous expression in Escherichia coli, chemical reconstitution of Fe/S clusters, cysteine mutagenesis, bioinformatic analysis and/or X-ray crystallography 12,13,30,31 . An important question is the relevance of the Fe/S cluster in the identified protein for the physiology of the original organism.…”

DNA charge transport as a first step in coordinating the detection of lesions by repair proteins

“…There is now evidence that mutations specifically in the iron-sulfur cluster domain of XPD result in TFIIH instability, thus leading to TTD (28,30). Similarly, mutations in the analogous region on the related FancJ gene (which also encodes a helicase with an iron-sulfur cluster) causes predisposition to early onset breast cancer (25,32,33). We have demonstrated that the [4Fe-4S] cluster in Sulfolobus acidocaldarius (Sa)XPD is redox-active, with a redox potential equivalent to that of BER proteins EndoIII and MutY [approximately 80 mV vs. normal hydrogen electrode (NHE)] (34).…”