Delving into the Choreography of DNA Repair

Photo: Coriell Institute for Medical Research COLD STORAGE: Cells from Coriell Institute for Medical Research that have been collected by and distributed to scientists from around the world, have played a key role in advancing the understanding of DNA repair. If early conceptions of DNA repair could be characterized as a dance, the process essential for maintaining genomic stability could be portrayed as an enzyme-to-enzyme pas de deux. However, a more accurate understanding of this DNA repair model in development now requires a more complex molecular choreography--an elaborate Broadway production whose players include genetics, structural biology, and signal transduction.1 "Our goal is to take our understanding of the individual excision enzymes to the next level [and] understand not only enzyme structure, DNA damage recognition, and catalysis for individual steps, but also how the enzyme and DNA conformational changes and timing allow directed channeling of damaged DNA; in other words, [to understand] the coordinated partner exchange of the enzymes within and between DNA repair pathways," notes John A. Tainer, professor of molecular biology at The Scripps Research Institute of La Jolla, Calif. "We are aiming to envision the molecular choreography of DNA-based damage repair--how the actions, movements, steps, conformational changes, partners, and timing are controlled by the nature of the DNA damage plus the structural chemistry of the participating enzymes." While Kenneth H. Kraemer, a research scientist at the National Cancer Institute, agrees that the DNA repair field is headed in that more unified--albeit more intricate--direction, he insists that the stage must first be set. "Before it's going there, we have to understand where it is," Kraemer comments. The foundation includes understanding the genes and gene products involved in xeroderma pigmentosum (XP), a genetic disorder that affects approximately one in a million people worldwide. People with that disorder have mutations in both copies of one of seven genes, labeled XP A through G. Those genes are essential for nucleotide excision repair (XP variant, a less understood type of xeroderma, shows reduced activity in another DNA repair system). "Most of [the XP genes] have been cloned and now we're trying to understand the functioning of their products." Patients who have defects in those genes can have a variety of clinical symptoms. Phenotypes include XP alone--meaning 1,000 times the cancer risk of the general public, XP with neurological disease, XP along with Cockayne syndrome, or trichothiodystrophy where patients have brittle hair, but, mysteriously, no increased cancer risk. "We're only just beginning to relate the mutations in the genes to the variations in the clinical symptoms," agrees James E. Cleaver, professor of dermatology at the University of California at San Francisco, who identified the DNA repair disorder in 1968. "You get some idea that the location of the mutation is related to the particular set of clinical symptoms that develop." Robert Johnson Robert Johnson, director of the Coriell Cell Repositories in Camden, N.J., which contains cell and DNA samples from every known variant of XP (see related story, below), notes that most of the individual parts of the DNA repair machinery have been identified. "Some of them are DNA damage-binding proteins, others are part of a molecular scissors complex that [is] able to cut aside part of the lesion. Others lift it out. Other proteins are involved in directing the repair machinery to damaged regions of active genes." While these parts may be known, their control and regulation are not well understood yet. "There are a lot of uncertainties about how the overall process is established on damaged chromatin," he comments. "One of the remaining problems is how does this enormous complex of proteins organize and change the nature of chromatin to allow the machine to work properly?" Perhaps more specifically, DNA repair researchers wonder how the cell's repair machinery performs triage--deciding to fix transcriptionally active portions of DNA first. "That ordering process requires a lot of regulation," notes Johnson. Kraemer adds that most of the focus thus far has been on active genes; transcriptionally active genes represent only a fraction of all genes involved in repair. Kraemer's lab is now trying to identify the range of phenotypes in XPC complementation groups, a type of XP more common in the United States. Kraemer notes that XPC seems especially fascinating because defects in that gene appear to affect repair of nontranscribed regions of DNA. Jin-an Feng Priscilla K. Cooper, a researcher in the Life Sciences Division, Department of Radiation Biology and DNA Repair at Lawrence Berkeley National Laboratory in Berkeley, Calif., suspects transcription's role in DNA repair could be related to other processes. In one paper, she showed that in XPG, transcription-coupled repair and base-excision repair are linked through the use of common enzymes that form repair machinery.2 "Probably all of the repair mechanisms function through large, multiprotein complexes," Cooper notes. Tainer, who calls Cooper's 1997 paper "pioneering," suspects that similar relationships will be discovered between other repair pathways. "These pathways are going to be coupled in all sorts of interesting ways," predicts Tainer. Cooper's work shows that some repair pathways pick and choose from a common toolbox of protein components, Johnson notes. "The switching of one machine into another, or the modification of one machine into another is one of the great things that cells are able to do." XPD also shares the dual role of DNA repair and transcription factor, notes Kraemer. "They may represent a link between repair and transcription, but we don't know exactly how it's working." He suspects that the structure--and related function--of each of those two proteins may help explain the relationship. "Both of those proteins function as helicases that unwind DNA. And the unwinding of the region of DNA is important if you're going to make RNA from it or if you're trying to find a lesion in the DNA and repair it. So you can see how that capacity of the helicase can be important in transcription and repair." Cooper notes that the events that initiate repair remain the most difficult to understand. Tainer and colleagues suspect that the complexes get their cue to assemble from the structure of DNA. "We're trying to see how the structure of the DNA damage and the structure of the enzyme control repair." Commonalities in the structures of several repair enzymes that have been characterized through X-ray crystallography may play a role.3,4,5,6,7,8 "These enzymes seem to have a lot of different folds, but they all seem to work the same way," Tainer comments. He suspects that they all recognize an extra helical flip. "We're beginning to envision how the initiating steps may be coordinated." Cooper and Tainer agree that signal transduction may provide an initiating event. Merging the fields of structural biology and signal transduction will be difficult but essential, they agree. Errol C. Friedberg Richard Fishel, professor of genetics and molecular biology at the Kimmel Cancer Center at Thomas Jefferson University in Philadelphia, is attempting to bring the two fields together. "I think the old way of thinking about this was in terms of complexes that did single repair events. The way that we think about it is entirely in terms of signaling. Only instead of signaling through the membrane, from the surface, and between cells, we're signaling on the DNA, and we're saying, 'There's something wrong with some place on this DNA. Let's take care of it.'" Fishel characterizes DNA repair as a set of well-ordered events, starting with recognition of damage and leading to building complexes of proteins that repair the lesion. Adenine nucleotide-bound proteins act as the "on" and "off" switches in this process, he suggests.9 Other groups have begun forming their own signal transduction links to DNA repair. Cleaver and colleagues recently suggested that the tumor suppressor gene p53 is linked to greater susceptibility to ultraviolet (UV) damage in the XP variant cells.10 Errol C. Friedberg, professor of pathology at University of Texas Southwestern Medical School at Dallas, is also examining p53 in animals with defects in DNA repair. Friedberg notes that the p53 protein plays a critical role in regulating the progression of cells through the cell cycle when they have been exposed to DNA damage. "Somehow--we don't know exactly how--various types of DNA damage signal the cell to halt progression through the cycle," Friedberg comments. A common, but not universally accepted notion, is that this arrest of cell cycle progression provides more time for cells to repair damaged DNA. P53 plays a role in sensing the presence of damage, then transducing this signal to the DNA repair machinery, Friedberg suspects. "My own studies have shown that in mice that are defective in one of the XP genes and hence are highly sensitive to UV radiation-induced skin cancer, simultaneous mutation of the p53 gene increases that cancer susceptibility," he explains. Other labs have made similar observations and have begun making other links from transduction to repair. A University of Tokyo team suggests an interaction between the XPB protein and the BCR-ABL oncogene.11 Although Kraemer cautions that such research is preliminary, he believes links between signal transduction and DNA repair will strengthen: "A lot of that is very exploratory now," he comments. "And that's where the research is going." Cleaver predicts that the links between structural biology and signal transduction will become stronger in time. "Understanding the complete structural phenomenon of how a damage site is actually recognized and acts as a signal, is a rapidly developing field." Jin-an Feng, a structural biologist at Fox Chase Cancer Center in Philadelphia, suggests that the problem must attract researchers from multiple disciplines. Feng and colleagues are studying the specificity between repair proteins and a damaged region of DNA. "We're particularly interested in how this recognition process is perceived, Feng notes. He adds that understanding such problems--and the complete process of the DNA repair events--will require more interdisciplinary research, since the process encompasses genetics, biochemistry, and structural biology. "We will get a rational understanding of a system much quicker," Feng concludes. S.S. Parikh et al., "Envisioning the molecular choreography of DNA base-excision repair," Current Opinion in Structural Biology, 9:37-47, February 1999. P.K. Cooper et al., "Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G," Science, 275:990-3, 1997. B. Shen et al., "Flap endonuclease homologues in archaebacteria exist as independent proteins," Trends in Biochemical Sciences, 23:171-3, May 1998. S.S. Parikh et al., "Base-excision repair initiation revealed by crystal structures and DNA-binding kinetics of human uracil-DNA glycosylase bound to DNA," EMBO Journal, 17:5214-26, September 1998. D.J. Hosfield et al., "Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human FEN-1," Journal of Biological Chemistry, 273:27154-61, October 1998. D.J. Hosfield et al., "Crystal structures of the DNA repair and replication exo- and endonuclease FEN-1: implications for coupling DNA repair and PCNA binding to enzymatic activity," Cell, 95:135-46, October 1998. Y. Guan et al., "Crystal structures of MutY: extra-helical adenine defines the specificity pocket and DNA binding orientation for the HhH DNA repair superfamily," Nature Structural Biology, 5:1058-64, December 1998. C.D. Putnam et al., "Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with E. coli uracil-DNA glycosylase," Journal of Molecular Biology, 287:331-46, March 1999. R. Fishel, "Mismatch repair, molecular switches, and signal transduction," Genes and Development, 12:2096-101, July 1998. J.E. Cleaver et al., "Increased ultraviolet sensitivity and chromosomal instability related to p53 function in the xeroderma pigmentosum variant," Cancer Research, 1102-8, March 1999. N. Takeda et al., "The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein," Proceedings of the National Academy of Sciences, 96:203-7, Jan. 5, 1999.

Delving into the Choreography of DNA Repair

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