<p>Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. </p><p>In chapter 1, the solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR are reported, revealing an atypical four-helix bundle. Yeast two-hybrid assays were used to identify a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR-binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. The combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes.</p><p>The multifaceted abilities of the Rev1 CTD are further explicated in chapter 2 where the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ RIR is reported. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a mega translesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.</p><p>Chapter 3 explores the ubiquitin-binding capacity of the FAAP20 UBZ in an effort to begin understanding its requirement for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery through recognition of monoubiquitinated Rev1. FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, it is revealed that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin-binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.</p><p>Having structurally elucidated the molecular basis of the essential scaffolding function of the Rev1 CTD, the search for small molecule inhibitors of the Rev1-Rev7 interaction has been initiated toward the goal of developing novel adjuvants to DNA targeting chemotherapeutics. Screening efforts have led to the discovery of a lead compound, JH-RE-06*NaOH, that specifically targets the Rev7-binding hydrophobic pocket of the Rev1 CTD with low micromolar affinity, effectively inhibiting the Rev1-Rev7 interaction in an in vitro ELISA assay developed for high-throughput screening of small molecule libraries. With the potential for positive outcomes in future in vivo assays, we hope to develop JH-RE-06*NaOH into the first potent inhibitor of translesion synthesis in cancer patients being treated with DNA-targetng chemotherapeutics to aid in sensitization and prevention of chemoresistance development in malignancies.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/9936 |
| Date | January 2015 |
| Creators | Wojtaszek, Jessica Louise |
| Contributors | Zhou, Pei |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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