Investigators say that knowing more about the mechanisms of these diseases may provide insights into how therapeutic drugs can be designed. All of the disorders occur due to inherited defects in a crucial DNA repair enzyme, the XPD helicase, which unwinds DNA to fix damage that regularly occurs.
In an article in the May 30, 2008 issue of the journal Cell, the researchers describe how they "built" the first crystal structure of the enzyme, and how that led them to see defects in the function of the protein that help explain these diseases. Li Fan of the Scripps Molecular Biology Department performed the x-ray crystallography of XPD at DOE's Stanford Linear Accelerator Center and the Advanced Light Source at Berkeley Lab. The biochemistry of XPD was assessed by Jill Fuss, a biochemist in Priscilla Cooper's laboratory in the Genome Stability Department of Berkeley Lab's LSD, and by Quen Cheng, a Research Associate in the Cooper lab.
"It was never understood why mutations in this gene, including some changes in amino acids that sit right next to each other, could produce such dissimilar disorders", stated the study's first author, Li Fan, a senior research associate in the laboratory of Scripps Research Professor John Tainer. "But now we can see problems that range from the inability of the enzyme to bind on to DNA to the tendency for it to get stuck while doing its job, and each issue produces different physical consequences."
Researchers at the Lawrence Berkeley National Laboratory and the San Diego Supercomputer Center also participated in the study. "The results from the combined biochemical and structural experiments were like turning on a light in a dark room and suddenly seeing for the first time how XPD - a key piece of machinery needed to open DNA to make proteins or to repair the DNA - was really working", John Tainer stated. "Besides its own biological importance, XPD is a paradigm for understanding how small, single site defects in one gene can cause such different outcomes in humans as aging, where too many cells die, and cancer, where mutants cells do not die but grow to become types of cancer."
DNA requires constant repair due to ongoing damage from the sun's ultraviolet (UV) rays, as well as from toxic chemicals and other insults. One primary way of doing that is through nucleotide excision repair, in which teams of enzymes recognize the damage, unwind the DNA helical structure, cut out mutated bases, and stitch the structure back together. The role of the XPD enzyme is to help find DNA damage and to unwind the double-stranded DNA at the lesion so the damaged DNA can be accessible to other DNA repair factors.
Surprisingly, mutations in the XPD gene are linked to three different inherited syndromes: Xeroderma pigmentosum (XP), which increases risk of a developing skin cancer by several thousand-fold; and Cockayne syndrome (CS) and trichothiodystrophy (TTD), both of which are premature aging and developmental disorders.
To conduct this study, researchers cloned the XPD gene - which is conserved across different species - from the single-cell organism, Archaea, and then expressed it in bacteria to obtain enough of the enzyme to crystallize it. Then the team mapped structural locations of the disease-causing mutations they knew existed on the human XPD gene - areas where one amino acid was substituted for another. "By doing that we could analyse the potential effect of the mutation on the structure, and predict the effects that would have on the function of the enzyme", Li Fan stated.
The scientists then made 16 different enzymes, each with one of the known mutations, and checked their predictions by actually testing these mutated enzymes in the laboratory for their biochemical functions. John Tainer noted: "Li Fan, Andy Arvia, and I worked with Jill Fuss and Quen Change at Lawrence Berkeley National Lab to characterize the normal and mutant XPD proteins for three different activities: ATPase activity - the ability to hydrolyze ATP for energy, DNA binding, and helicase activity - the ability to unwind double-stand DNA."
The combination of these three different biochemical activities with the detailed structure from x-ray crystallography solved some of the mysteries regarding how small changes in the XPD sequence could cause the different human diseases.
Two versions of the protein were needed to do x-ray crystallography: the native conformation and a variant in which all of its methionine amino acid residues were replaced by selenomethionines. In the synchrotron's beam the heavy selenium atoms signal their presence and allow unambiguous registration of the protein's methionines in proper sequence.
The structure of XPD came as a surprise. Of the protein's four domains - distinct substructures, two are helicase motifs, HD1 and HD2; another is the iron-sulfur domain, 4FeS, which protrudes in a way that implies an important role in opening DNA and sensing DNA damage. The fourth domain, called the Arch, was wholly unexpected. With 4FeS, the Arch domain forms a curious archway over a tunnel-like opening at the end of a long groove. Given the electric charge of the amino acids lining the groove, the topograpy suggests a passageway for channeling DNA.
"If you stretched out the linear sequence of the protein, you would never guess that those different pieces fold together", stated Priscilla Cooper. Proteins result from folding linear strings of amino acid residues, but in XPD the folding is an intricate knot. One of the helicase domains, HD1, takes portions of its sequence from separate regions of the linear string, and the Arch and 4FeS domains sprout from different parts of HD1.
The complex topography of the protein, and the specific amino acid residues of which it is formed, suggest the specialized functions of different structural features. The helicase domains are designed for flexible movement; the channels seem well suited to binding DNA, or the ATP molecules that when hydrolyzed provide the energy for unwinding; and the oxidation-sensitive 4FeS domain is strategically placed for detecting DNA damage.
The team found that the five different mutations that lead to the XP disorder either reduce activity of the XPD enzyme or disable its function altogether. That means damage caused by UV light cannot be efficiently repaired. Li Fan stated: "If you can't unwind DNA to repair the damage, it accumulates, leading to exponential increases in development of skin cancer."
Mutations responsible for XP/CS, a disorder that combines both CS and XP, "are more dramatically severe", according to Li Fan. "The enzyme is not only unable to repair damage, it gets hung on the DNA it is trying to unwind, and stops." Blocking DNA in this way prevents other molecules known as transcription factors from reading the genes "downstream" of the stuck XP enzyme, leading to a premature aging syndrome and mental retardation in children who inherit the defective gene, along with increased sun sensitivity.
"XPD mutations that lead to TTD fundamentally disrupt the framework of the XPD protein", Li Fan stated. "It changes the stability of the enzyme, and may not allow it to bind to DNA or to other proteins. We think it affects transcription of genes that are responsible for later stages of cell differentiation, such as the molecules that control production of hair and fingernails."
"We now have a molecular basis for distinguishing these diseases", Li Fan stated, "and we can use these models to help shed more light on these childhood disorders." In addition to Li Fan and John Tainer, authors of the study, titled "XPD Helicase Structures and Activities Provide Insights into the Cancer and Aging Phenotypes from XPD Mutations", were Andrew Arvai from Scripps Research; Victoria Roberts from the San Diego Supercomputer Center; and Jill O. Fuss, Quen J. Cheng, Michal Hammel, and Priscilla K. Cooper from Lawrence Berkeley National Laboratory. This work was supported by grants from the National Institutes of Health and the United States Department of Energy, as well as by the Skaggs Institute for Chemical Biology at Scripps Research.
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