Advanced search options

Advanced Search Options 🞨

Browse by author name (“Author name starts with…”).

Find ETDs with:

in
/  
in
/  
in
/  
in

Written in Published in Earliest date Latest date

Sorted by

Results per page:

Sorted by: relevance · author · university · dateNew search

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

Search Limiters

Last 2 Years | English Only

No search limiters apply to these results.

▼ Search Limiters


University of Colorado

1. Zinder, John Charles. In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1.

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

Chromosomal DNA replication is an essential and demanding task that all cells must undertake in order to proceed with division. Decades of research have made Escherichia coli (Eco) the prototype for the study of DNA replication and many of the lessons learned from the study of the Eco replisome, the term for the proteins that carries out replication, have held true in other organisms. DNA replication in Bacillus subtilis (Bsu), a bacterium that diverged from Eco approximately 1.5 billion years ago, follows many of the same general principles of Eco with the important difference being that it uses two DNA polymerases rather than one. One polymerase, PolC, performs leading strand synthesis as well as the majority of lagging strand synthesis while the other polymerase, DnaE, is responsible for only a short extension of an RNA primer at the lagging strand before handing off synthesis to PolC to finish the Okazaki fragment. The details of this handoff have been examined using in vitro primer extension assays. It was found that DnaE is sufficiently processive that it must be actively evicted from the primer terminus in order for the exchange to occur. Additionally, in vitro studies of the replisome of phage SPP1, a virulent dsDNA phage that infects Bsu, have been performed. It was found that a phage encoded ssDNA binding protein was able to inhibit the PolC polymerase in the context of the host replisome but not the phage replisome, providing a novel mechanism through which a phage can subvert the host replication machinery while ensuring use of its own. These studies expand on the already rich repertoire of known strategies for cellular and viral DNA replication, and lay the way for more detailed studies of their precise mechanism. Advisors/Committee Members: Charles S. McHenry, Robert T. Batey, Marcelo C. Sousa.

Subjects/Keywords: Bacillus subtilis; DnaE; DNA replication; PolC; Polymerase exchange; SPP1; Biochemistry; Structural Biology

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

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

APA (6th Edition):

Zinder, J. C. (2013). In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1. (Masters Thesis). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/108

Chicago Manual of Style (16th Edition):

Zinder, John Charles. “In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1.” 2013. Masters Thesis, University of Colorado. Accessed January 26, 2020. https://scholar.colorado.edu/chem_gradetds/108.

MLA Handbook (7th Edition):

Zinder, John Charles. “In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1.” 2013. Web. 26 Jan 2020.

Vancouver:

Zinder JC. In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1. [Internet] [Masters thesis]. University of Colorado; 2013. [cited 2020 Jan 26]. Available from: https://scholar.colorado.edu/chem_gradetds/108.

Council of Science Editors:

Zinder JC. In vitro Studies of DNA Replication in Bacillus subtilis and Phage SPP1. [Masters Thesis]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/108


University of Colorado

2. Manhart, Carol Michelle. Investigating protein-DNA interactions at replication forks by photo-crosslinking.

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

During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and trigger release from the lagging strand. A hypothesis in the literature proposes that the τ subunit (of the DnaX complex) directly senses completion of an Okazaki fragment. An alternative model suggests that the polymerase subunit senses conversion of a gap to a nick. I show using a novel phenyldiazirine photo-crosslinker linked to the 5-position of thymidylate that the τ subunit is not in position to distinguish gapped DNA from nicked DNA. The α subunit (the polymerase) is positioned to serve as the processivity sensor. Upon encountering duplex DNA, the polymerase likely changes conformation triggering its release from the lagging strand and the β processivity clamp, modulating its own affinity. Unrepaired replication forks dissociate from the helicase and suffer collapse. PriA recognizes stalled replication forks and initiates interactions to reload the helicase and activate a previously stalled fork. I used a FRET helicase assay to develop a PriA- dependent helicase loading system in E. coli and B. subtilis and to identify a minimal substrate to support a photo-crosslinking study also discussed here. I discovered that PriA's ATPase activity dictates substrate specificity. I also show that PriA serves as a checkpoint protein by blocking the replicase from binding to stalled replication forks distinguishing between an alternative model. SPP1 is a bacteriophage that infects B. subtilis. It encodes its own initiation proteins (origin binding protein, primosomal proteins, helicase, and single-strand binding protein (SSB)) but requires its host's primase and major replicative polymerase to replicate its genome. Both host and phage SSBs can support a reconstituted SPP1 system, but phage SSB does not support a reconstituted B. subtilis system. Using the B. subtilis FRET helicase assay, I show that phage SSB can substitute for the host's SSB in helicase reloading. Therefore the defect in the reconstituted system is not at the level of helicase loading or function and must occur after the helicase is loaded. I also show an absolute requirement on all SPP1 components in helicase reloading, including the origin binding protein (in a non-origin-containing template), which suggests a new role for this protein. In collaboration with Tim Lohman's lab at Washington University, I have contributed to a study into the functions of the C-terminal tails of SSB. SSB functions as a homotetramer whose four C-terminal tails interact with many other proteins necessary for DNA replication and repair. In an in vivo assay, an SSB variant that has two functional C-terminal tails supports viability in E. coli. An SSB variant that has one C- terminal tail is dominant lethal. In a reconstituted rolling circle E. coli replication system, there is a defect in coupled synthesis that causes a two-fold decrease in lagging strand synthesis relative to the leading strand using the variant… Advisors/Committee Members: Charles S. McHenry, Robert Kuchta, Robert Batey, Marcelo Sousa, Thomas Perkins.

Subjects/Keywords: Biochemistry

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

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

APA (6th Edition):

Manhart, C. M. (2013). Investigating protein-DNA interactions at replication forks by photo-crosslinking. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/78

Chicago Manual of Style (16th Edition):

Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Doctoral Dissertation, University of Colorado. Accessed January 26, 2020. https://scholar.colorado.edu/chem_gradetds/78.

MLA Handbook (7th Edition):

Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Web. 26 Jan 2020.

Vancouver:

Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Jan 26]. Available from: https://scholar.colorado.edu/chem_gradetds/78.

Council of Science Editors:

Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/78


University of Colorado

3. Jansen, Katarina Bartoš. Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis.

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

Gram-negative bacteria are characterized by the presence of an outer membrane (OM), which is a highly selective permeability barrier that protects the bacteria from the environment and allows for exchange of nutrients and waste products. The OM consists of phospholipids located in the inner leaflet and lipopolysaccharide located in the outer leaflet of the membrane, as well as embedded β-barrel proteins and lipoproteins facing the periplasmic side. Integral outer membrane protein (OMP) biogenesis is essential making it an ideal antibiotic target. The molecular mechanisms that mediate OMP biogenesis are still poorly understood. It has been established that the machinery responsible for β-barrel protein assembly into the OM consists of the OMP BamA and four lipoproteins (BamB-E). It is not clear yet how the β-Barrel Assembly Machinery (BAM) complex folds OMPs and/or inserts them in the OM. We approached this problem through the structure-function analysis of this system. Here, I present the crystal structure of BamB from Pseudomonas aeruginosa and a structure of BamA-BamB chimera protein from Escherichia coli. Using these structures we were guided to test some plausible functions of the proteins on the path to uncover the BAM mechanism during the folding and insertion of OMPs into the OM. BamB is an eight-bladed β-propeller with conserved two loops protruding from the bottom of the propeller. The loop cluster residues previously shown to mediate BamB-BamA interaction are located at POTRA3 near the flexible hinge between POTRA2 and 3 region of BamA. These structures are a valuable guide to improve our understanding of BAM complex mechanism. Advisors/Committee Members: Marcelo C. Sousa, Joseph J. Falke, James Goodrich, Charles S. McHenry, Michael H.B. Stowell.

Subjects/Keywords: beta-barrel assembly machinery; membrane proteins; outer membrane; structural biology; Biochemistry; Biophysics; Molecular Biology

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

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

APA (6th Edition):

Jansen, K. B. (2013). Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/103

Chicago Manual of Style (16th Edition):

Jansen, Katarina Bartoš. “Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis.” 2013. Doctoral Dissertation, University of Colorado. Accessed January 26, 2020. https://scholar.colorado.edu/chem_gradetds/103.

MLA Handbook (7th Edition):

Jansen, Katarina Bartoš. “Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis.” 2013. Web. 26 Jan 2020.

Vancouver:

Jansen KB. Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Jan 26]. Available from: https://scholar.colorado.edu/chem_gradetds/103.

Council of Science Editors:

Jansen KB. Probing the Structure and Function of the β-barrel Assembly Machinery Responsible for Outer Membrane Protein Biogenesis. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/103

.