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Title: Structure and mechanism of a uracil-DNA glycolase essential for γ-herpesviral DNA maintenance and replication
Author: Earl, Christopher
ISNI:       0000 0004 6352 9964
Awarding Body: Birkbeck, University of London
Current Institution: Birkbeck (University of London)
Date of Award: 2017
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Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus belonging to the -herpesvirus subfamily. KSHV is the etiologic agent of all forms of Kaposi's sarcoma (KS) and has been linked with the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman's disease. Effcient -herpesvirus DNA replication requires a virally encoded uracil-DNA glycosylase (UNG) as a structural element of the viral replisome. This replication-associated role of viral UNG is independent of UNG substrate catalysis but is mediated by the DNA binding region of UNG. The UNG leucine loop, essential for DNA binding, is embellished in all -herpesvirus UNGs by a 7-residue insertion which is highly conserved in length but not in sequence. To investigate the effects of this insertion on UNG-DNA binding and its potential role in the viral replisome, a crystal structure of KSHV UNG (kUNG) was solved in its product complex with duplex DNA. The kUNG-DNA structure captures a novel DNA conformation induced by the apparently structurally conserved leucine loop insertion. As seen in other structures, the substrate uracil has been processed having been ipped out of the DNA base stack and into the kUNG active site. Unusually though, the orphaned partner base of the substrate uracil is also ipped out of the DNA duplex by kUNG. The orphan base is solvent-exposed and presented in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more widely open than in other UNG-DNA structures. Protein engineering reveals that ipping out of the orphan base is not required for enzymatic activity and mutagenesis studies suggest a model in which the leucine loop is pinned out of the -herpesvirus UNG DNA binding cleft until DNA contact induces insertion of the loop into the duplex to effect orphan base- ipping.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available