1. Volume 23, Issue 6, Pages 801-808 (September 2006)
The DNA Repair Helicases XPD and FancJ Have Essential Iron-Sulfur Domains 
Jana Rudolf, Vasso Makrantoni, W. John Ingledew, Michael J.R. Stark, Malcolm F. White 
Molecular Cell 
Volume 23, Issue 6, Pages (September 2006)
DOI: /j.molcel
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2. Figure 1 A Conserved Metal Binding Domain in the XPD Protein Family
(A) Schematic of the human XPD protein, with the canonical helicase motifs indicated by black boxes and the position of the Fe-S domain shown in green.
(B) Sequence alignment of the region between the Walker A and B boxes (helicase motifs I and II), including the Fe-S domain, for a variety of human and archaeal helicases. Sac, Sulfolobus acidocaldarius XPD; Pfu, Pyrococcus furiosus XPD; Rad3, Saccharomyces cerevisiae Rad3; XPD, human XPD; RTel1, human RTel1; FancJ, human FancJ/BACH1; and Chl1, human Chl1. The four conserved cysteine residues are highlighted in yellow. The positions of the clinically relevant XPD mutation R112H and the FancJ mutation A349P are highlighted in blue.
(C) Structure of the UvrB helicase, showing the β hairpin domain in blue interacting with a bound DNA species in red (Truglio et al., 2006).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Archaeal XPD Contains an Fe-S Cluster
(A) Visible absorbance spectrum of pure S. acidocaldarius XPD showing the shoulder of absorbance at 400 nm for the wild-type (WT) protein that is absent in the K84H, F136P, and C137S mutants. The inset shows the color of a 150 μM solution of WT XPD protein.
(B) Electron paramagnetic resonance (EPR) spectrum of oxidant-treated XPD. X band EPR spectrum of the oxidized (ferricyanide-treated) enzyme. The spectrum is characteristic of an oxidized [3Fe-4S] Fe-S cluster. EPR conditions: temperature, 30 K; modulation frequency, 100 KHz; modulation amplitude, 0.32 mT; microwave frequency, 9.49 GHz; and microwave power, 0.2 mW.
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4. Figure 3 ssDNA-Dependent ATPase Activity of XPD
(A) Rate of ATP hydrolysis by WT and two mutant forms of XPD in the presence of double- and single-stranded phiX174 DNA. WT (black circles), C88S (red triangles), and K84H (blue squares) XPD proteins were assayed in the presence of ssDNA (filled symbols) and dsDNA (open symbols). Data points are the means of triplicate measurements, with standard errors of the mean (SEM) indicated.
(B) Temperature dependence of the ssDNA-dependent ATPase activity of WT XPD. Reactions contained 50 nM XPD and 5 nM of a 70 nt oligonucleotide. SEM obtained from triplicate data points are indicated.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Helicase Activities of XPD Mutants
(A) Schematic of the helicase assay and substrate design.
(B) WT XPD is an ATP-dependent DNA helicase. The displaced DNA strand accumulates over time upon incubation at 45°C. There is no strand displacement in the absence of ATP or XPD.
(C) Helicase activity of WT and mutant forms of XPD. The K35A control mutant is inactive as a helicase but has a stable Fe-S cluster. For the four cysteine mutants, helicase activity correlates with the presence of a stable Fe-S domain. Mutagenesis of SacXPD to mimic the clinically relevant XPD mutation R112H and FancJ mutation A349P destabilizes the Fe-S cluster and abolishes helicase activity. The presence or absence of a stable Fe-S cluster is indicated by a green or gray circle, respectively.
(D) Quantification of the helicase activity of WT and mutant XPD enzymes at 45°C. Each data point represents the mean of triplicate measurements, with SEM indicated. The data were fitted with a smooth curve.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Mutations of the Yeast Rad3p Fe-S Domain Confer a UV-Sensitive Phenotype
Equivalent 10-fold serial dilutions of haploid yeast strains containing the indicated WT or mutant copies of RAD3 were spotted onto YPD agar plates, irradiated with the indicated flux of ultraviolet light, and photographed after 2 days growth at 26°C in the dark. Control, no irradiation. Equivalent residue numbers in SacXPD and Rad3 are K35 and K48, K84 and R111, and C88 and C115, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions