By Alexandria Oviatt
A recent Nucleic Acids Research paper from the lab of Walter Chazin (Biochemistry) reveals key interactions in a DNA repair pathway called nucleotide excision repair (NER) that may have implications for the treatment of the genetic disorder xeroderma pigmentosum (XP). Individuals burdened with this disease have mutations in some of the proteins necessary for NER that impair the DNA repair response and result in hypersensitivity to sunlight and a more than 2,000-fold increase in the risk of skin cancer.
The NER pathway requires the precise assembly of at least 20 different proteins at bulky DNA lesions to cut out the damaged portion, fill in with the original DNA sequence, and restore the genetic material to its original state. Two of those proteins, XPA and RPA, have been shown to function as a scaffold to help properly position the proteins that remove the damage over the DNA. Previous studies from the Chazin group had already defined an interaction between one end of XPA and a flexible portion of RPA, but new nuclear magnetic resonance (NMR) and X-ray scattering data allowed the group to model the dynamic binding of the two proteins and to characterize a second interaction between their DNA-binding regions.
RPA binds to the intact DNA strand to protect it from the action of the other proteins, but which orientation XPA takes with respect to the DNA lesion has been a matter of controversy. The Chazin group proposes a model in which XPA binds to either side of the damage, allowing its DNA-binding domain to engage both RPA and the DNA. In turn, this allows both proteins to interface and form stabilizing contacts with each other while remaining bound to the DNA.
The Chazin lab tested their model using XPA-deficient cell extracts to determine the importance of XPA-RPA binding in the NER pathway. When researchers added wild-type XPA to damaged DNA in the extracts, what they observed suggested that the DNA had been completely repaired. However, when XPA carrying mutations in its DNA-binding domain (where RPA also binds) was added back into the XPA-deficient cells, NER was clearly impaired and the DNA remained damaged; mutations in XPA did not prevent DNA binding.
This research provides support for the Chazin model by showing that the interaction between the DNA-binding domains of XPA and RPA may be more important for the positioning of the NER scaffold proteins compared to the previously characterized interaction of their flexible domains.
Characterizing the interactions between these two key NER proteins not only lays to rest a controversy in the field, but may provide a starting point in the design of small molecules that could target these interactions and provide a therapeutic avenue for XP patients.
Funding for this work has been provided by the National Institutes of Health, the Korean Institute for Basic Science, the National Science Foundation, the U.S. Department of Energy, and Vanderbilt University.