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The iron-containing domain is essential in Rad3 helicases for coupling of ATP hydrolysis to DNA translocation and for targeting the helicase to the single-stranded DNA-double-stranded DNA junction
Journal article   Open access   Peer reviewed

The iron-containing domain is essential in Rad3 helicases for coupling of ATP hydrolysis to DNA translocation and for targeting the helicase to the single-stranded DNA-double-stranded DNA junction

Robert A Pugh, Masayoshi Honda, Haley Leesley, Alvin Thomas, Yuyen Lin, Mark J Nilges, Isaac K O Cann and Maria Spies
The Journal of biological chemistry, Vol.283(3), pp.1732-1743
01/18/2008
DOI: 10.1074/jbc.M707064200
PMID: 18029358
url
https://doi.org/10.1074/jbc.M707064200View
Published (Version of record) Open Access

Abstract

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.
 Archaeal Proteins - metabolism Protein Structure, Tertiary DNA Helicases - chemistry DNA, Single-Stranded - metabolism Archaeal Proteins - chemistry Adenosine Triphosphatases - metabolism DNA, Cruciform - metabolism Substrate Specificity DNA Replication Mutant Proteins - metabolism Structure-Activity Relationship Mutation - genetics Archaea - enzymology Hydrolysis Thermodynamics Biological Transport Adenosine Triphosphate - metabolism Protein Binding Ligands Iron-Sulfur Proteins - metabolism Amino Acid Substitution

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