Scripps Research Crystal Structure Reveals Mystery behind Three Rare Childhood Disorders
News May 30, 2008
Researchers at The Scripps Research Institute have figured out how it is that tiny mutations in a single gene can produce three strikingly different childhood diseases - disorders that increase cancer risk thousands of times in some young patients and premature aging or a complete failure to develop in others.
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.
"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," says 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," Tainer said.
"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 analyze the potential effect of the mutation on the structure, and predict the effects that would have on the function of the enzyme," Fan says.
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.
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.
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. Fan says, "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 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," Fan says. "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," Fan says, "and we can use these models to help shed more light on these childhood disorders."
Source of a Potent Greenhouse Gas IdentifiedNews
A study of a Lake Erie wetland suggests that scientists have vastly underestimated the number of places methane-producing microbes can survive—and, as a result, today’s global climate models may be misjudging the amount of methane being released into the atmosphere.READ MORE
New Cell-weighing Technique Helps Predict How Drugs Affect Cancer CellsNews
Researchers at MIT have now shown that they can use a new type of measurement to predict how drugs will affect cancer cells taken from multiple-myeloma patients.READ MORE
Researchers Discover Mutation That Appears to Protect Against Multiple Aspects of Biological AgingNews
The first genetic mutation that appears to protect against multiple aspects of biological aging in humans has been discovered in an extended family of Old Order Amish living in the vicinity of Berne, Indiana, report Northwestern Medicine scientists.READ MORE