Structural and Functional Characterization of the Human DNA Repair Helicase XPD by Comparative Molecular Modeling and Site-directed Mutagenesis of the Bacterial Repair Protein UvrB

Abstract

A molecular model for the human nucleotide excision repair protein, XPD, was developed based on the structural and functional relationship of the protein with a bacterial nucleotide excision repair (NER) protein, UvrB. Whereas XPD does not share significant sequence identity with UvrB, the proteins share seven highly conserved helicase motifs that define a common protein structural template. They also have similar functional roles in their ATPase activity and the ability to unwind DNA and verify damaged strands in the process of NER. The validity of using the crystal structure of UvrB as a template for the development of an XPD model was tested by mimicking human disease-causing mutations (XPD: R112H, D234N, R601L) in UvrB (E110R, D338N, R506A) and by mutating two highly conserved residues (XPD, His-237 and Asp-609; UvrB, H341A and D510A). The XPD structural model can be employed in understanding the molecular mechanism of XPD human disease causing mutations. The value of this XPD model demonstrates the generalized approach for the prediction of the structure of a mammalian protein based on the crystal structure of a structurally and functionally related bacterial protein sharing extremely low sequence identity (<15%). Nucleotide excision repair (NER)1 is a process by which damaged nucleotides, from UV light and chemical carcinogens, are removed from DNA. NER is one of the most highly conserved biochemical pathways, and enzymes that mediate this process appear in prokaryotes, archaea, and eukaryotes (1, 2). NER can be viewed in the following five continuous steps: 1) damage recognition and verification, 2) incision, 3) excision, 4) repair synthesis, and 5) ligation. During the damage recognition step, a protein complex first identifies a structural perturbation in the helical DNA, which is then verified by additional damage processing and strand opening proteins. Once the lesion has been verified, endonucleases are recruited to the damaged strand performing the initial incision 3Ј followed by a 5Ј incision facilitating the excision of an oligonucleotide containing the damage. Repair synthesis fills in the resulting gap. Finally DNA ligase seals the newly completed repair patch. The evolutionary conservation of proteins involved in NER is exemplified by human XPD, which maintains 56 and 52% (protein) sequence identity, respectively, with the equivalent Schizosaccharomyces pombe, Rad15, and Saccharomyces cerevisiae, RAD3, proteins over the length of the entire protein (BLAST alignments). Human and mouse XPD retain 98% (protein) sequence identity (3).XPD is one of the constituent proteins forming the TFIIH complex that is responsible for damage processing and strand verification (4, 5). XPD and XPB have intrinsic helicase activities that are absolutely required for NER (4). TFIIH is also required for initiation of RNA polymerase II transcription.

Structural Insights into the Cryptic DNA-dependent ATPase Activity of UvrB

“…Interestingly, the equivalent Asp338Asn mutation in XPD, the human analogue of UvrB, has been found to be highly prevalent amongst xeroderma pigmentosum patients. Based on the molecular modelling studies described by Bienstock et al, 22 we also note that Lys67, invariant amongst the bacterial UvrB homologues, is replaced by threonine (Thr76) in XPD. A Thr76Ala mutation has been found in xeroderma pigmentosum sufferers, presenting the possibility that there may be functional conservation of this residue where the hydroxyl group of Thr76 could mediate interactions similar to those of the sidechain N z moiety of Lys67 in UvrB.…”

“…Arg543 has been shown to be an essential residue in repair, since its mutation to histidine results in a protein that is repair-deficient, exhibiting a loss of helicase activity and ATPase stimulation by DNA in the presence of UvrA. 22 Although its role has been speculated upon, from our structure it would appear that Arg543 functions to stabilise the product and possibly substrate complexes.…”

Analyzing the Handoff of DNA from UvrA to UvrB Utilizing DNA-Protein Photoaffinity Labeling

“…We have shown previously that the D510A UvrB mutant is defective in UvrA⅐DNA damagedependent ATPase activity leading to a loss of incision efficiency (43). This mutant does not form a UvrB⅐DNA complex as determined by EMSA (Fig.…”

Meta-analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer

“…The two polymorphisms have shown little or no effect on the protein using predictive models [4] or evolutionary analysis [5]. Structural evidence indicated that these polymorphisms are located outside the main catalytic sites [6] and far from regulatory [7] and interacting domains [8] suggesting that they have no direct effect on the ATPase activity of ERCC2. Additional studies detected no measurable effect of the two variants on NER capacity and basal transcription activation [2], and genotype-specific differences in DNA repair rates was inconsistent [9].…”