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Title:XPD Helicase: Shifting the Inchworm Into Reverse
Author(s):Pugh, Robert A.
Doctoral Committee Chair(s):Spies, Maria
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:Directional translocation by helicases results in duplex separation and displacement of bound proteins which allows for the DNA processing events associated with DNA repair, replication, recombination, and transcription. Unresolved questions regarding DNA helicases include: (1) how is directional translocation determined in SF2 helicases; (2) do helicases use the mechanism for unwinding as they use for translocation; (3) the role of unique insertions found among an otherwise conserved motor core; and (4) how these enzymes function as integral members of larger DNA processing machines. Here, I used the archaeal XPD helicase to answer these four fundamental questions. This work characterizes XPD as a 5' to 3' ssDNA translocase that preferentially unwinds forked DNA substrates, contains an FeS cluster, binds to the ssDNA-dsDNA junction of forked substrates, and is capable of unwinding forked substrates up to 40 bps as a monomer in the presence of RPA2. I found the unique insertion in XPD that harbors an FeS cluster was responsible for targeting the helicase to the ssDNA-dsDNA junction, properly orienting the helicase for unwinding, and coupling of ATP hydrolysis with translocation. How human XPD functions as part of the TFIIH complex in nucleotide excision repair was also explored by looking at unwinding in the presence of the ssDNA binding protein, RPA, a known component in NER. This work also is the first to provide evidence for the orientation of a SF2B helicase bound to DNA, and shows evidence for a secondary DNA binding domain on XPD which contacts the displaced strand during duplex unwinding.
Issue Date:2009
Description:191 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
Other Identifier(s):(UMI)AAI3392438
Date Available in IDEALS:2014-12-17
Date Deposited:2009

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