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You searched for +publisher:"University of Colorado" +contributor:("Charles McHenry"). Showing records 1 – 3 of 3 total matches.

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University of Colorado

1. Lund, Travis John. Asymmetric Recognition of Nucleobase Features by DNA Polymerases.

Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado

This work describes an investigation into the recognition of nucleobase features by several DNA polymerases. I used of a series of pyrimidine analogues modified at O2, N-3, and N4/O4 to determine how the Klenow fragment of DNA polymerase I, an A family polymerase, and two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether or not to polymerize pyrimidine dNTPs. Removal of these heteroatoms generally impaired polymerization, with the effects varying from mild to severe. Removing O2 of a pyrimidine dNTP vastly decreased incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O2 from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N4/O4 greatly impaired polymerization, both of the resulting dNTP analogues as well as polymerization of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Removing O2 from a pyrimidine at the primer 3'-terminus also prohibited extension of the primer. Importantly, these studies indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. I also describe initial work on the synthesis of a novel dibasic analogue incorporating chemical features whose importance has been demonstrated in this work. These features include the presence of minor groove hydrogen bond acceptors to facilitate extension past the analogue upon its incorporation, as well as the Watson-Crick hydrogen bonding groups of an A:T pair, since we have seen that the removal or modification of these groups has unpredictable, but often detrimental, effects upon efficient nucleobase incorporation by polymerases. Advisors/Committee Members: Robert Kuchta, Jennifer Kugel, Charles McHenry, Byron Purse, Amy Palmer.

Subjects/Keywords: analogue; DNA; fidelity; kinetics; nucleotide; Polymerase; Biochemistry; Organic Chemistry

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APA (6th Edition):

Lund, T. J. (2013). Asymmetric Recognition of Nucleobase Features by DNA Polymerases. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/75

Chicago Manual of Style (16th Edition):

Lund, Travis John. “Asymmetric Recognition of Nucleobase Features by DNA Polymerases.” 2013. Doctoral Dissertation, University of Colorado. Accessed April 01, 2020. https://scholar.colorado.edu/chem_gradetds/75.

MLA Handbook (7th Edition):

Lund, Travis John. “Asymmetric Recognition of Nucleobase Features by DNA Polymerases.” 2013. Web. 01 Apr 2020.

Vancouver:

Lund TJ. Asymmetric Recognition of Nucleobase Features by DNA Polymerases. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Apr 01]. Available from: https://scholar.colorado.edu/chem_gradetds/75.

Council of Science Editors:

Lund TJ. Asymmetric Recognition of Nucleobase Features by DNA Polymerases. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/75


University of Colorado

2. Yuan, Quan. Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork.

Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado

The E. coli chromosome is replicated by a dimeric DNA polymerase III holoenzyme (Pol III HE) in a reaction where continuous leading and discontinuous lagging strand synthesis are coupled. Two models have been proposed to depict how a lagging strand polymerase dissociates from the preceding Okazaki fragment and cycle to the next primer. The collision model proposes that the polymerase collides with the 5'-end of the preceding Okazaki fragment and triggers release, whereas the signaling model suggests that the polymerase is signaled to cycle by synthesis of a new primer by primase. I developed a mini-circle DNA replication system with a highly asymmetric G:C distribution between DNA strands to differentiate these models. Specific perturbations of lagging strand synthesis by incorporation of ddGTP (chain termination) or dGDPNP (decreased elongation rate) on dCMP-containing lagging strand template confirm the signaling model and rule out the collision model. The lagging strand polymerase elongates much faster than the leading strand polymerase, explaining why gaps between Okazaki fragments are not found under physiological conditions. The presence of a primer, not primase, provides the signal to trigger cycling. Full-length Okazaki fragments (in the presence of dNTPs) and equivalent gaps between fragments (in the presence of dGDPNP) were obtained using reconstituted E. coli replicase regardless of the number of the τ DnaX subunits present in the clamp loader. I characterized an intrinsic helicase-independent strand displacement activity of the DNA Pol III HE and found that Pol III is stabilized by an interaction with SSB on the displaced strand by a Pol III-τ-ψ-χ-SSB interaction network. PriA, the initiator of replication restart on stalled replication forks, blocks the displacement reaction. E. coli SSB functions as a homotetramer with each subunit possessing a C-terminus interacting with other proteins that function in DNA replication and repair. To assess how many C-termini of SSB are required for function in DNA replication, I carried out rolling circle DNA replication assays using concatemeric forms of SSB that possess only one or two C-termini. I discovered that SSB "tetramers" with one C-terminus cause a decrease in DNA synthesis and uncouples leading and lagging strand synthesis. Advisors/Committee Members: Charles McHenry, Robert Kuchta.

Subjects/Keywords: Biochemistry

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Yuan, Q. (2013). Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/91

Chicago Manual of Style (16th Edition):

Yuan, Quan. “Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork.” 2013. Doctoral Dissertation, University of Colorado. Accessed April 01, 2020. https://scholar.colorado.edu/chem_gradetds/91.

MLA Handbook (7th Edition):

Yuan, Quan. “Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork.” 2013. Web. 01 Apr 2020.

Vancouver:

Yuan Q. Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Apr 01]. Available from: https://scholar.colorado.edu/chem_gradetds/91.

Council of Science Editors:

Yuan Q. Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/91


University of Colorado

3. Dickey, Thayne Henderson. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.

Degree: PhD, Chemistry & Biochemistry, 2014, University of Colorado

Telomere dysfunction has been implicated in several diseases including cancer and aging. The two main roles of telomeres are often defined as end-protection and length regulation and the shelterin complex lies at the heart of both of these functions. Pot1 is the single-stranded DNA-binding component of shelterin and is important for genome stability, telomere length regulation, and C-strand resection, all functions that rely on its ssDNA-binding ability. In the work presented here, we structurally and biochemically characterize the dual OB-fold DNA-binding domain of Pot1 from the model organism S. pombe. This work provides insight into potential mechanisms of telomere length regulation and elucidates novel and broadly applicable features of protein/nucleic acid recognition. X-ray crystal structures of the second OB-fold of Pot1, Pot1pC, bound to various ssDNA sequences reveal a unique plasticity at the DNA-binding interface. Global rearrangements of the entire interface allow the accommodation of complementary base substitutions despite the abundance of apparently base-specific hydrogen bonds. This structural plasticity likely explains the ability of S. pombe Pot1 to accommodate the natural heterogeneity in S. pombe telomeric sequence. Furthermore, these mechanisms of accommodation allow for a high-affinity/low-specificity binding mode that is likely utilized by other sequence nonspecific ssDNA and ssRNA-binding proteins. We also use NMR and biochemical techniques to study how the behavior of the individual OB-folds is modulated in the context of the complete DNA-binding domain. Multiple DNA-binding domains often exist within a single protein or complex in biology, but the functional importance of these tandem arrangements is rarely known. The work described here reveals a malleable interface between domains that allows for multiple DNA-binding modes. These binding modes have differing structural and biochemical features that may be important for telomere length regulation. Advisors/Committee Members: Deborah S. Wuttke, Robert T. Batey, Marcelo C. Sousa, Loren E. Hough, Charles McHenry.

Subjects/Keywords: Plasticity; Pombe; Pot1; protein nucleic acid; shelterin; telomere; Biochemistry; Molecular Genetics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Dickey, T. H. (2014). Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/111

Chicago Manual of Style (16th Edition):

Dickey, Thayne Henderson. “Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.” 2014. Doctoral Dissertation, University of Colorado. Accessed April 01, 2020. https://scholar.colorado.edu/chem_gradetds/111.

MLA Handbook (7th Edition):

Dickey, Thayne Henderson. “Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1.” 2014. Web. 01 Apr 2020.

Vancouver:

Dickey TH. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. [Internet] [Doctoral dissertation]. University of Colorado; 2014. [cited 2020 Apr 01]. Available from: https://scholar.colorado.edu/chem_gradetds/111.

Council of Science Editors:

Dickey TH. Structural Plasticity in the Recognition of ssDNA by the Telomeric Protein Pot1. [Doctoral Dissertation]. University of Colorado; 2014. Available from: https://scholar.colorado.edu/chem_gradetds/111

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