Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item MODULATION OF HIV-1 REVERSE TRANSCRIPTASE AND FAMILY A DNA POLYMERASE PRIMER-TEMPLATE BINDING(2014) Fenstermacher, Katherine Joan; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polymerases are enzymes used by all cellular and viral organisms to replicate their genomes. The human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT), uses a single-stranded RNA template to create double-stranded DNA during the course of the viral life cycle. Successful reverse transcription relies on the speed of catalysis and the ability of the enzyme to stay bound to the template during synthesis. I demonstrate that both of these properties can be modulated by the presence of different divalent cations, fundamentally altering the behavior of HIV RT. In the presence of 2 mM Mg2+, the HIV RT primer-template complex has a half-life of 1.7±1.0 min, incorporating nucleotides at a maximum rate of 3.5 nucleotides (nt) per second (average speed 1.4±0.4 nt/sec). Substituting 2 mM Mg2+ with 400 μM Zn2+ dramatically slows the speed of catalysis (maximum 0.1 nt/sec, average 0.022±0.003 nt/sec) and promotes primer-template complexes that last hours (half-life of 220±60 min). These profound changes to the enzyme's function critically inhibit reverse transcription, even in the presence of optimal concentrations of Mg2+. In addition to the cation composition during reverse transcription, previous studies have demonstrated that the sequence of the primer-template substrate can also affect the duration of a RT-primer-template complex. In light of this discovery, I investigated the tendency of two Family A DNA polymerases, the Thermus aquaticus DNA polymerase (Taq pol) and the Klenow fragment from Escherichia coli DNA polymerase I (Klenow), to selectively and tightly bind primer-template complexes. Using Primer-Template Systematic Evolution of Ligands by Exponential Enrichment (PT SELEX), I determined that both Taq pol and Klenow tightly bind to sequences containing regions that match the initiation and melting domains of promoters for the structurally similar bacteriophage T7-like RNA polymerases. This suggests a shared sequence preference that might be present in all Family A DNA polymerases, derived from a common ancestor. I plan to exploit this primer-template binding preference to advance biotechnologies utilizing these enzymes.Item Nucleocapsid protein modulates the specificity of plus strand priming and recombination patterns in Human Immunodeficiency Virus(2008-11-30) Jacob, Deena Thankam; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Replication in HIV (human immunodeficiency virus) occurs through reverse transcription in which the genomic single stranded RNA is copied into double stranded DNA. This process involves two priming events namely those of the minus and plus strand DNAs. The tRNA primer required to initiate the minus strand is carried by the virus into the host cell, while the plus strand primer is generated from a region of the genomic RNA called the polypurine tract (PPT). Results in this dissertation indicate a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. For HIV, the central and 3′ (PPTs) are the major sites of plus strand initiation and other primers are rarely used. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Extension assays in presence of mutant NCs show that the helix destabilization activity of NC and its ability to block the association of RT to non-PPT primers are responsible for the preferential extension of PPT in presence of NC. The effect of varying NC and Mg2+ concentrations on recombination during reverse transcription was also analyzed in this thesis. NC strongly influences the efficiency of recombination as well as the location where crossovers occurred. In contrast Mg2+ had a smaller effect on crossover locations. Both NC and Mg2+ influenced the level of pausing by RT during synthesis on RNA templates although NC's effect was more profound. At high NC concentrations, pausing was nearly eliminated even in locations with high predicted secondary structure. The results suggest that RT pausing may be limited during virus replication.