The genetic information which is the fundamental of organism is coded as base sequence in the double-stranded DNA(dsDNA). DNA is constantly damaged by various environmental factors such as ultraviolet light, radiation, and toxic substances. Organism develops DNA repair system to restore the damaged DNA (*1). Nucleotide excision repair(NER) is one of such mechanism by which the damaged nucleotides was detached from the DNA. And then, the resulting gap is filled by DNA polymerase. In prokaryotes, this processes are conducted by Uvr protein complex which consists of four subunits: UvrA, UvrB, UvrC, and UvrD. (For Uvr complex, refer to PDB:2FDC). Although the fundamental processes are similar, NER has more complexity in eukaryotes or ancient bacteria. It is known that protein complex TFIIH which consists of 9 subunits involves the eukaryotic NER. XPD, one of the subunit of TFIIH, has ATP-dependent helicase activity, which split damaged dsDNA segment into two single-strand DNAs(ssDNAs). This protein also have a role of stabilizing the interactions among other TFIIH subunits. Therefore, XPD can be regarded as a essential molecule for NER system. It is known that the point mutations in the human XPD gene (ERCC2) cause three different genetic disorders: Xeroderma Pigmentosum(XP), Cockayne Syndrome combined with XP (XP/CS) and Trichothiodystrophy(TTD). Although these three diseases share a common photosensitivity phenotype, they differ greatly in their predispositions to cancer or accelerated aging. For example, XP patients show several 1000-fold increase in skin cancer, whereas neither CS nor TTD patients show an increase in the cancer incidence despite sun sensitivity. Interestingly, mutations which are very close together, even in adjacent amino acids, can cause different diseases. In human XPD, for instance, a mutation in R601L causes XP; A mutation in G602D causes XP/CS (Fig.3). To address the question how point mutations in adjacent residues in a single enzyme can give rise to such different disease phenotypes, the crystal structure of XPD derived from Sulfolobus acidocaldarius was determined and analyzed.
(*1) There are several types of DNA repair mechanisms, as follows.
The structure of Sulfolobus acidocaldarius XPD (SaXPD) consists of four domains : HD1, HD2, 4FeS, and Arch domain. (Fig.1). The two helicase domains, HD1 and HD2, contain six conserved helicase motifs. The ATP-binding site of XPD is lie in the cleft formed between the two domains. 4FeS domain is likely to involve with sensing DNA damage. It was confirmed that the 4Fe-4S cluster that combined with the 4FeS domain has a role of maintaining the overall stability of the enzyme. Arch domain forms two gates by interacting with 4FeS and HD2 domain respectively. The 50-angstrom long channel extending along the helicase motifs in HD2 is gated at both ends by the two gates. This channel provides a passageway for ssDNA translocation.
The DNA-tube shown in the Fig.1 and Fig.2 was superimposed by using known helicase-DNA complex structures (2P6R.pdb) as a template.
Compared with the human XPD with 761 residues, the SaXPD, composed of 551 residues, lacks the C-terminal extension domain (CTE). However, most of the point mutation sites which are known to cause genetic disorders in human XPD are contained in the four domains (Fig.3).
The crystal structure of SaXPD has shown the relationship between XPD mutation sites and its function. First, most of the XP causing mutation sites (shown in red sphere in Fig.2 and red flag in Fig.3) are lie along the DNA-binding channel. These residues must involve with the DNA-binding activity of XPD. Second, the XP/CS raising mutation sites (shown in yellow sphere in Fig.2, and yellow flag in Fig.3) tend to exist at the ATP-binding site. This observation suggests that these residues contribute to HD1-HD2 conformational change, which is needed to the helicase activity. Third, the TTD-related mutation sites (shown in blue sphere in Fig.2 and blue flag in Fig.3) are exist in all four domains. Mutations at these sites are expected to result in framework defects that decrease the TFIIH integrity. These structural observations provide significant information to understand the relationship between XPD mutations and disease consequences.
Protein Data Bank (PDB)
Fan, L. Fuss, J.O. Cheng, Q.J. Arvai, A.S. Hammel, M. Roberts, V.A. Cooper, P.K. Tainer, J.A.; "XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations"; Cell(Cambridge,Mass.); (2008) 133:789-800.
author: Jun-ichi Ito