Structural Biology: DNA Repair and Structurebased Drug Design - Caroline Kisker
E-mail: caroline.kisker@virchow.uni-wuerzburg.de Phone: +49(0)931 201 483 00 Fax: +49(0)931 201 487 02 http://www.rudolf-virchow-zentrum.de/forschung/kisker.html
It has been shown that 80 to 90% of all human cancers are due to DNA damage. Among the various DNA repair mechanisms available to the cell nucleotide excision repair (NER) stands out because of its broad substrate specificity. This system recognizes damages such as the carcinogenic pyrimidine dimers induced by UV radiation, benzo[a]pyrene-guanine adducts caused by smoking, as well as guanine-cisplatinum adducts formed during chemotherapy. We aim to understand the fundamental mechanisms of the bacterial and mammalian NER machinery. Since damage can accumulate and may not be repaired prior to replication and due to the role of DNA polymerases in certain diseases, we also analyze different DNA polymerases and their role in genetic maintenance. A second focus is structure-based drug design to identify new therapeutics against infectious diseases. Our main targets are essential bacterial enzymes involved in fatty acid biosynthesis.
Nucleotide Excision Repair The importance of this repair mechanism is reflected by three severe inherited diseases in humans that are due to defects in NER: xeroderma pigmentosum, Cockayne’s syndrome and trichothiodystrophy. We use structural, biochemical and biophysical methods to characterize the individual components of NER and their cognate complexes, which are vital to the reaction cascade. Three major aspects in the “recognition” and “repair” events of NER are still not understood: (1) What are the structural determinants of the DNA required for damage recognition? (2) How are damage-induced conformational changes in the DNA perceived by a DNA repair protein complex, and how does the recognition of proteinDNA contacts translate into high binding affinities? (3) How does the recognition process lead to incision? 5’ Incision of the damage containing oligonucleotide Although the proteins involved are different, eukaryotic and prokaryotic cells share the same basic mechanism of recognition and incision, making the well-characterized bacterial proteins an ideal target for structural analysis. NER in bacteria, one of the first repair mechanisms discovered, is mediated by the products of the uvrA, uvrB and uvrC genes. The proteins UvrA, UvrB and UvrC recognize and cleave the damaged DNA in an ATP-dependent, multi-step reaction. After the damage has been identified, UvrA dissociates, while UvrB remains bound to the DNA in a stable pre-incision complex. UvrC binds to this complex and triggers the incision four nucleotides 3’ to the damaged site, followed by an incision seven nucleotides 5’ to the damaged site. This cascade of events and the involvement of several proteins in damage recognition the catalytic domain responsible for 5’ incision, which is followed by two helixhairpin-helix motifs. We recently solved the crystal structure of this C-terminal half of UvrC. Despite the lack of sequence homology the endonuclease domain shares surprising structural homology to the RNase H fold.
Fig. 1: A: Domain architecture of UvrC; B: Structure of the C-terminal half of UvrC.
and repair ensure discrimination between damaged and non-damaged DNA. UvrC has been shown to catalyze both the 3’ and the 5’ incisions by two distinct catalytic sites that can be inactivated independently. The C-terminal half of UvrC contains
The fold of the endonuclease domain and a similar active site is also found in other enzymes with nuclease or polynucleotide transferase activity and has been identified in retroviral integrases, the PIWI domain of Argonaute, DNA transposases and RNase
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HII. The active site in these proteins contains usually a highly conserved carboxylate triad. In UvrC, however, the triad is composed of two aspartates and one histidine and is extended to a tetrad by a lysine, which has been shown to be critical for catalysis as well. The helix-hairpin-helix (HhH) motifs mediate protein-DNA contacts, which are critical for the incision reaction. We solved several structures of the C-terminal half of UvrC, thus providing several snapshots of the protein. The position and orientation of the helix-hairpin-helix motifs relative to the endonuclease domain varies significantly in these structures, which is achieved through a flexible linker between both domains. This flexibility is necessary to position UvrC correctly for the incision reactions.
the solvent. The EM model for the pol γ holoenzyme provides considerable insight into the structure and function of the polymerase, explaining features of this enzyme that distinguish it from other family A DNA polymerases. Our EM model also permits us to predict how known DNA binding domains of pol γB may help to guide the path of DNA along the holoenzyme in a way that enhances the processivity of the polymerase, but may impair its fidelity. Extramural Funding DFG (SFB 630, TP B7), (KI-562/2-1) NIH R01 GM070873 NCI 5PO1 1CAO4799514
Selected Publications Karakas, E., Truglio, JJ., Croteau, D., Rhau, B., Wang, L., Van Houten, B., and Kisker, C. (2007) Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad. EMBO J, 26, 613-622. Zwahlen, J., Kolappan, S., Zhou, R., Kisker, C., and Tonge, PJ. (2007) Structure and Mechanism of MbtI, the Salicylate Synthase from Mycobacterium tuberculosis. Biochemistry, 46, 954-964. Kolappan, S., Zwahlen, J., Zhou, R., Truglio, JJ., Tonge, PJ., and Kisker, C. (2007) Lysine 190 is the catalytic base in MenF, the menaquinone-specific isochorismate Synthase from Escherichia coli: Implications for an enzyme family. Biochemistry, 46, 946-953. Tonge, P.J., Kisker, C., and Slayden, R.A. (2007) Development of Modern InhA Inhibitors to combat drug resistant strains of Mycobacterium tuberculosis. Curr Top Med Chem, 7, 489-498. Yakubovskaya, E., Lukin, M., Chen, Z., Berriman, J., Wall, JS., Kobayashi, R., Kisker, C., and Bogenhagen, DF. (2007) The EM structure of human DNA polymerase γ reveals a localized contact between the catalytic and accessory subunits. EMBO J, 26, 42834291. Kisker, C. (2007) When one protein does the job of many. Structure, 15, 1163-1165.
Fig. 3: Subunit localization in pol γ, yellow: pol γA, blue and red: pol γB.
Structure based Drug Design We recently solved the first structure of the enoyl -ACP- reductase FabI from Francisella tularensis, the organism causing tularemia. This structure will serve as the basis to analyze conformational changes that will be induced through different diphenyl ethers that were previously developed in the laboratory of our collaborator against the homologous enzyme in Mycobacterium tuberculosis. The goal is the identification of a slow onset inhibitor to obtain promising in vivo inhibition.
Fig. 2: A: Different orientations of the HhH motifs in UvrC ; B: Model of UvrC interacting with DNA.
Human DNA Polymerase γ DNA polymerase γ is not only essential for mitochondrial DNA replication and repair, but also the target of a number of antiviral compounds such as the nucleoside analogues azidothymidine and dideoxynucleotides employed in HIV therapy, which lead to severe neuromuscular toxicity. We used electron microscopy to examine the structure of human DNA pol γ, the heterotrimeric mtDNA replicase, containing one pol γA and two accessory pol γB subunits, implicated in certain mitochondrial diseases and aging models. Our analysis permitted unambiguous identification of the position of the accessory factor within the holoenzyme. One pol γB subunit dominates contacts with the catalytic subunit while the second B subunit is largely exposed to
Fig. 4: Structure of FabI from Francisella tularensis.
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