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

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-doublestranded 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.The function of many multisubunit DNA repair complexes requires activity of DNA helicases, which are ubiquitous, highly diverse molecular motors that convert the chemical energy of ATP binding and hydrolysis into mechanical work of unidirectional translocation along the DNA lattice (reviewed in Refs. 1, 2). Unidirectional translocation of a helicase may be coupled to other thermodynamically unfavorable processes, including separation of the nucleic acid duplexes and disassembly of protein-nucleic acid complexes. Coupling of ATP hydrolysis to translocation is achieved through the set of so-called "helicase signature motifs" that define the motor core of the enzyme (3, 4). The motor cores of numerous helicases are structurally and mechanistically similar, yet these enzymes display remarkable functional diversity (for review see Ref. 4). It has become clear in recent years that such diversity may be achieved in trans through utilization of specific processivity factors or in cis by incorporating additional structural features that direct interaction with nucleic acids, duplex destabilization, and strand separation activities.The Rad3 (XPD) 2 helica...

Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

“…Recently, a 4Fe-4S cofactor was discovered in p58C and disruption of the cluster by mutation of conserved cysteine residues was shown to abolish primase activity (14,15). Several lines of evidence suggest that iron-sulfur clusters are essential for the function of many types of DNA processing proteins, but the specific roles of these essential cofactors have not been elucidated (16)(17)(18). Hence, even though the importance of the cluster has been established, the role of p58C in initiation of DNA replication remains poorly understood, primarily due to a lack of information about its structure and binding partners.…”

The Association of the Xeroderma Pigmentosum Group D DNA Helicase (XPD) with Transcription Factor IIH Is Regulated by the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…The XPD homologue from Sulfolobus acidocaldarius was shown to bind a Fe-S cluster that was not required for either its global stability or its singlestranded DNA binding and ATPase activities but was essential for its in vitro helicase activity (7). Pugh et al (8) showed that integrity of Fe-S cluster on FacRad3 (Ferroplasma acidarmanus) was necessary for the folding and structural stability of an auxiliary domain required for coupling ATP hydrolysis and translocation activity. X-ray crystallography studies of XPD revealed that the Fe-S cluster was bound to a unique insert embedded within helicase domains 1 and 2 (9 -11) and could potentially function by separating duplex DNA at the singlestranded DNA-double-stranded DNA junction.…”

The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication

“…The specific roles of Fe/S clusters include electron transport, sensing oxidative stress, detecting DNA damage, stabilizing protein structure, forming enzymatic active sites, and functioning as regulatory cofactors (24,29,53). Fe/S cluster domains have also been proposed to act as part of a molecular "plowshare" in unwinding DNA duplex to support the function of many DNA helicases (30)(31)(32)(54)(55)(56). We therefore set out to examine the possible physiological significance of MCPyV sT Fe/S clusters.…”

“…Fe/S cluster domains play critical roles in many cellular nucleic acid processing enzymes, including those involved in DNA unwinding, endonuclease activity, reduction of ribonucleotides to deoxyribonucleotides, and DNA polymerization (24,(27)(28)(29)(30)(31)(32)(33). In light of the observation that MCPyV sT stimulates LT viral replication (18,19), we tested how mutating the Fe/S cluster-coordinating cysteines in MCPyV sT affects its function in viral DNA replication.…”