Enzyme crystal structure reveals 'unexpected' genome repair functions

New discovery could help improve some forms of chemotherapy

The study is being published in an advance online version of the journal Molecular Cell.

The research looked at XPB helicase from an archaea, a single cell organism similar to bacteria. Helicases are enzymes that unwind or separate the strands of the nucleic acid double helix, an action that is critical to transcription and nucleotide excision repair (NER), as well as other cell processes.

"XPB was initially identified as the gene responsible for NER defects in xeroderma pigmentosum patients, who are hypersensitive to light and have a dramatically increased risk of skin cancer," says John A. Tainer, a professor at Scripps Research and its Skaggs Institute for Chemical Biology who led the study. "This reflects the fact that XPB plays a key role in unwinding damaged DNA during NER, which removes a broad spectrum of DNA lesions, including those caused by exposure to ultraviolet light."

DNA needs constant repair because of the damage from a variety of sources that occurs to its base pairs of nucleotides. It is estimated that in every human cell more than 10,000 DNA bases are repaired each day, making NER critically important for cell survival and protection against mutations. NER is a critical defense mechanism that removes DNA lesions caused by the mutating effects of sunlight (ultraviolet light) and toxic chemicals.

In addition, NER is critical to the success of the anticancer drug cisplatin, since cisplatin works by initiating the process of DNA repair, in turn activating apoptosis or programmed cell death when the repair process fails. "Because chemotherapeutic agents like the chemotherapy drug cisplatin and radiation therapy work by essentially damaging DNA, any new understanding of the DNA repair mechanism could mean potential improvements in the treatment of cancer," Tainer says.

Prior to this study, there were no specific models for how XPB acts in DNA separation either to initiate transcription or to begin NER. There were also no models for the role that XPB, which is an essential subunit of Transcription Factor IIH (TFIIH) functional assembly complex, might play in changing conformations for TFIIH's alternate roles in either transcription or DNA repair.

The XPB crystal structures developed by the researchers identified unexpected functional domains for XPB that, according to the study, help "address key questions about XPB structure-function relationships for transcription and nucleotide excision repair."

Research Associate Li Fan of Scripps Research, the first author of the study, adds, "We were surprised when we found that XPB contains a domain structurally similar to the mismatch recognition domain of a bacterial DNA repair protein MutS. MutS helps recognize and repair mismatched DNA in E. coli. These two proteins have little sequence similarity. Biochemical assays following this discovery indicate that this domain allows XPB to interact with damaged DNA and enhances its unwinding activity on damaged DNA."

The report suggests that unknown protein and DNA interactions at transcription sites activate XPB within the TFIIH complex to allow it to start the DNA unwinding process.

"Even though TFIIH does not act directly in initial damage recognition, the interaction of XPB with the DNA lesion suggests that XPB plays a role in switching TFIIH from transcription mode to NER," Tainer says. "The structural biochemistry of XPB that we discovered shows an unexpected molecular mechanism by which XPB plays a key role in determining exactly how TFIIH functions, whether in transcription or repair mode."

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Other authors of the paper include Andrew Arvai, Priscilla K. Cooper, Shigenori Iwai, and Fumio Hanaoka.

The study was supported by the Human Frontiers of Science Program, the National Cancer Institute, and the U.S. Department of Energy.

About The Scripps Research Institute
The Scripps Research Institute, headquartered in La Jolla, California, in 18 buildings on 40 acres overlooking the Pacific Ocean, is one of the world's largest independent, non-profit biomedical research organizations. It stands at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel.

Scripps Florida, a 364,000 square-foot, state-of-the-art biomedical research facility, will be built in Palm Beach County. The facility will focus on basic biomedical science, drug discovery, and technology development. Palm Beach County and the State of Florida have provided start-up economic packages for development, building, staffing, and equipping the campus. Scripps Florida now operates with approximately 160 scientists, technicians, and administrative staff at 40,000 square-foot lab facilities on the Florida Atlantic University campus in Jupiter.


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