Probing the dNTP Binding Region of <em>Bacillus subtilis</em>: DNA Polymerase III with Site-Directed Inhibitors: A Dissertation
AuthorsButler, Michelle Marie
Faculty AdvisorNeal C. Brown
Academic ProgramBiochemistry and Molecular Pharmacology
UMass Chan AffiliationsDepartment of Pharmacology
Document TypeDoctoral Dissertation
DNA Polymerase III
Nucleic Acids, Nucleotides, and Nucleosides
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Abstract6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below. Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor's aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP. Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor's aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor's aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/31428
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