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Biochemical and Structural Studies of AlkB: A 2-oxoglutarate Iron(II) Dependent Dioxygenase

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Biochemical and Structural Studies of AlkB: A 2-oxoglutarate Iron(II) Dependent Dioxygenase
Holland, Paul
Unrepaired DNA alkylation damage can be both cytotoxic and mutagenic to cells leading to the activation of apoptotic pathways and the development of cancer. The cytotoxic effects of DNA alkylating agents have been the focus of research and utilization of these compounds as chemotherapeutics for the treatment of cancers. To evade the effects of DNA alkylation damage, prokaryotes and eukaryotes activate DNA damage repair pathways that lead to the expression of various DNA damage repair enzymes. AlkB is a direct repair 2-oxoglutarate iron-II dependent dioxygenase that oxidizes alkyl lesions occurring on the N1 position of purines and the N3 position of pyrimidines in both DNA and RNA. AlkB preferentially repairs 1-methyladenine and 3-methylcytosine lesions, but it is capable of repairing 1-methylguanine, 3-methylthymine, and 1-N6ethenoadenine lesions albeit at a less efficient rate. The dioxygenase activities of AlkB have been conserved throughout prokaryotes and eukaryotes where there are up to nine homologs found in human cells. Structures of AlkB and AlkB homologue DNA complexes have validated many of the biochemical properties observed by these unique enzymes. However, there are many questions that remain regarding the substrate specificity and DNA binding properties of these enzymes. In order to understand structurally the mechanisms underlying substrate specificity and DNA binding, the crystal structure of a D135A mutant enzyme was determined to 2.2 Å in complex with a 17-mer oligonucleotide with a 1-methylguanine lesion bound to the active site. Also, an unliganded wild type AlkB structure was also determined to 2.9 Å. Comparisons of the nucleotide bound structure with the unliganded structure demonstrated that Y76 participates in a “gating action” that opens the substrate binding pocket for the methylated base to enter and upon binding it seals the substrate in the catalytic complex. Additionally, the DNA binding loop expands ~2.5 Å once DNA binds to allow the phosphodiester backbone to rest in the DNA binding groove. Structural comparison of the 1-methylguanine structure with 1-methyladenine structures demonstrate that AlkB binds both lesions identically, and it supports the biochemical data that the lower efficiency of AlkB for 1-methylguanine substrates is due to the selectivity that D135 imposes on DNA bases when they enter the binding pocket. Analysis of the current AlkB crystal structures permitted the development of mutagenesis strategies to investigate amino acid residues that may be involved in substrate binding and catalysis. In vitro activity and DNA binding assays were performed on each mutant. Data obtained from these experiments demonstrated that the substrate binding pocket in AlkB created by W69 and D135 is critical for maintaining the catalytic complex through stabilization of the base for catalysis to occur. Additionally, it was discovered that D135 holds the key to the specificity of this enzyme for DNA methylation damage on adenine and cytosine due to an exocyclic nitrogen present on these bases as opposed to guanine and thymine. In vitro activity assays with this mutant displayed no detectable activity on 1-methyladenine substrates, but its activity on 1-methylguanine substrates increased ~40-fold in comparison to the wild type enzyme. Also, equilibrium binding data using methylated and undamaged oligonucleotides provided evidence for AlkB searching each base for DNA damage in ssDNA substrates. Significant reduction in activity and DNA binding using a Y76A and T51A mutation suggested these residues also participate in substrate stabilization and DNA base searching through Y76 hydrogen bonding to the phosphate of the methylated base and T51 stabilizing the DNA binding loop which forms extensive contacts to the phosphodiester backbone. Recent studies have linked two human AlkB homologues, hABH8 and FTO, to human bladder cancer, growth and development, and obesity. Preliminary experiments have been performed on an AlkB homologue from Drosophila melanogaster (DmAlkB) that has 60% sequence identity to hABH8. This enzyme is unique in that it contains an AlkB domain and a methyltransferase domain. It is hypothesized that this enzyme may be involved in protein translation and regulation through modification of specific tRNAs. Nucleic acid precipitation assays suggest that DmAlkB primarily interacts with RNA. Additionally, the human FTO protein has been cloned and purified in the laboratory. Structural studies and biochemical studies are currently being carried out on these two enzymes. The data presented in this dissertation have provided valuable insight into the mechanisms of substrate specificity and DNA binding of the AlkB enzyme that can be applied to other studies on AlkB homologues and may assist in the development of inhibitors of these proteins to increase the efficacy of current chemotherapeutics.
Structural Biology
DNA repair
Alexander, Rebecca (committee chair)
Hollis, Thomas (committee member)
Lyles, Doug (committee member)
Perrino, Fred (committee member)
Lowther, W. Todd (committee member)
2009-12-02T18:21:48Z (accessioned)
2010-06-18T18:57:25Z (accessioned)
2009-12-02T18:21:48Z (available)
2010-06-18T18:57:25Z (available)
2009-12-02T18:21:48Z (issued)
Biochemistry & Molecular Biology (discipline)
http://hdl.handle.net/10339/14692 (uri)
en_US (iso)
Wake Forest University
Release the entire work for access only to the Wake Forest University system for one year from the date below. After one year, release the entire work for access worldwide. (accessRights)

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