Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Deborah S. Wuttke

Second Advisor

Robert T. Batey

Third Advisor

Thomas Cech

Fourth Advisor

Robert Kuchta

Fifth Advisor

Norman Pace

Abstract

Many critical cellular functions involve the management of single-stranded DNA (ssDNA). For the vast majority of these processes ssDNA is only transiently present. Telomeres, the ends of eukaryotic chromosomes, are a special case where ssDNA is a necessary feature. Telomeres consist of a suite of proteins associated with long tracts of repetitive GT-rich DNA terminating in a conserved 3′ ssDNA overhang. The protection and regulation of these overhangs requires the sequence-specific binding activity of the telomere-end protection (TEP) family of proteins. The telomeric DNA of Schizosaccharomyces pombe consists of a core repeat sequence (GGTTAC) interrupted by spacer elements of variable length and sequence. The ssDNA-binding activity of the S. pombe TEP (SpPot1) is conferred by a DNA-binding domain consisting of two subdomains, Pot1pN and Pot1pC. Previous work has shown that Pot1pN binds a single repeat of the core telomere sequence (GGTTAC) with exquisite specificity, while Pot1pC binds an extended sequence representing 1.5 repeats (GGTTACGGT) with only modest specificity. Fulllength SpPot1 binds the composite 15mer, (GGTTAC)2GGT, and a shorter two-repeat 12mer, (GGTTAC)2, with equally high affinity (< 3 pM), but with substantially different kinetic and thermodynamic properties. The binding mode of the SpPot1/15mer complex is more stable than that of the 12mer complex, with a 2-fold longer half-life and increased tolerance to nucleotide and amino acid substitutions. In addition, SpPot1 accommodates significant sequence alterations within the 3′ end of telomeric oligonucleotides. The data suggest that SpPot1 protection of heterogeneous telomeres is mediated through 5′ sequence recognition and the use of alternate binding modes to maintain high affinity interaction with the ssDNA overhang.

Disruption of the end-binding activity of SpPot1 provides an avenue for studying telomere uncapping. The rich chemical diversity manifest in SpPot1/ssDNA interaction suggested binding could be perturbed by small molecule inhibition (SMI). Presented here are the successful implementation of a high-throughput screen and the identification of a single inhibitor of SpPot1/ssDNA binding. The compound binds specifically to the ssDNA-binding surface and inhibits via specific trimerization of the protein. These results suggest the utilization of SMI of TEP proteins as a feasible tool for the continued analysis of telomere-end protection.

Included in

Biochemistry Commons

Share

COinS