Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation

Abstract

The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a short DNA fragment, thus revealing the polarity of the translocated strand and providing insights into how the enzyme achieves its 5'-3' directionality. Accompanied by a detailed mutational and biochemical analysis of taXPD, we define the path of the translocated DNA strand through the protein and identify amino acids that are critical for protein function.

Figure 1

The XPD–DNA complex. (A) Overall structure of XPD with the two RecA-like domains in yellow and red, the FeS cluster domain in cyan, and the arch domain in green. The DNA identified in the electron density is shown in orange. (B) Enlarged view showing the tetranucleotide visualized in the structure with its carbon atoms in light blue. Residues interacting with the DNA are shown with their carbon atoms in grey and hydrogen bonds are indicated by dashed green lines. (C) Side view of the taXPD–DNA complex. Colour scheme is similar to (A). The cleft where the DNA is bound is indicated with arrows. The additional N-terminal helix harbouring the Q-motif is shown in grey. (D) Combination of experimentally verified DNA (orange) with modelled DNA (grey). The colour scheme for XPD is as described above.

Figure 2

Putative phosphate positions in the binary XPD–DNA complex. (A) The first sulphate molecule is located in the ATP-binding pocket of the Walker A motif in HD1 and is shown in all bonds representation. Hydrogen bonds are indicated by dashed green lines. (B) The second sulphate molecule is located in close proximity to the FeS cluster, in the basic groove at the exit of the pore.

Figure 3

Model of the XPD–DNA complex and location of the XPD variants. (A) Model of the XPD–DNA complex using the apo-XPD structure with the protein shown in a transparent surface representation. The different variants are shown as CPK models in green, orange, and red, indicating the strength of reduction for DNA binding compared with the wild-type protein (red ∼450-fold reduction; orange ∼35-fold reduction; green ∼5-fold reduction (Y166 is an exception and displays only an ∼2.5-fold reduction) relative to the wild-type protein). The surface of the subdomains of XPD is coloured as follows: HD1 (yellow), HD2 (red), arch (green), and FeS (cyan). The modelled ssDNA is shown in a cartoon representation. Experimental ssDNA is shown with an orange backbone and the ssDNA model in black. (B) Close-up view into the area of HD2 where the cleft is located. (C) Residues located at the pore, or in close proximity to the pore, after rotation of the molecule by 180° as indicated by the arrow.

Figure 4

Helicase activity of taXPD and its variants. The activity of wild-type XPD and the variants is shown in a bar diagram. The inset shows a typical kinetic measurement; ND indicates that the activity was not detectable.

Figure 5

Different functionalities within the XPD protein. The surface representation of XPD and the four views highlight different possible functions of specific residues within the protein. The red area is located in HD2 and may play a role in ratcheting, whereas the yellow area depicts residues abolishing helicase activity. The green areas show residues generating a hyperactive helicase. The arrows indicate the location of important features discussed in the text. The two arrows for the basic groove denote the start and end point of the groove.