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Catalano, Carlos E Sanyal, Saurarshi Jyoti 2013-11-14T20:53:35Z 2015-12-14T17:55:48Z 2013-11-14 2013 Sanyal_washington_0250E_12247.pdf http://hdl.handle.net/1773/24142 Thesis (Ph.D.)--University of Washington, 2013 Packaging of viral genomes into procapsids by terminase enzymes is conserved in many DNA viruses. Terminases bind to linear concatemers of replicated viral genomes and concomitantly excise (mature) and package a single genome per procapsid. In this thesis, I interrogate the role of E. coli integration host factor (IHF) in mediating the site-specific assembly of bacteriophage lambda terminase at its cognate DNA site, cos, which serves as the packaging initiation site. IHF binds to an I-element within cos and introduces a strong bend in the duplex. It was previously demonstrated that the small terminase subunit could stabilize an IHF-induced bend at cos. I hypothesized that terminase holoenzyme and IHF cooperatively assemble at cos and wrap the duplex into a compact nucleoprotein complex. Rigorous analysis of this cooperative assembly is complex due to the multiple terminase and IHF binding elements within cos. Therefore, I dissected the cos site into individual specific and nonspecific IHF binding sequences, and analyzed the relevant protein affinities for these subsites as well as for (1) the full-length cos site and (2) a random nonspecific (NS) sequence of equivalent length. Analytical ultracentrifugation and electrophoretic mobility shift studies show that IHF and terminase only modestly discriminate between cos and NS-DNA substrates; however, the two proteins cooperatively bind to cos-DNA. The data suggest that IHF confers site-specificity of binding to terminase. Also evident is significant nonspecific DNA binding concurrent with specific interactions, even on specific DNA substrates. IHF likely facilitates the high-affinity cooperative assembly of a relevant nucleoprotein complex at the cos site despite significant nonspecific binding of both proteins to DNA, with the functional significance of nonspecific DNA binding being the enhancement of protein-DNA interactions. Furthermore, sedimentation equilibrium studies demonstrate that while terminase assembles in the absence of IHF as a dimer on a 274 bp DNA substrate inclusive of the entire cos site, in the presence of IHF a nucleoprotein complex of mass consistent with five terminase protomers and two IHF molecules results. This finding further implicates IHF in the cooperative assembly of a specific ternary IHF-DNA-terminase complex at the packaging initiation site of bacteriophage lambda. A terminase packaging enzyme that both (1) site-specifically matures DNA and (2) packages DNA in a sequence-independent manner must be capable of both specific and nonspecific DNA binding. This work furthers the understanding of (1) one of the factors (IHF) involved in the site-specific assembly of a nucleoprotein complex required for the initiation of viral packaging, and (2) the nature of the specific nucleoprotein complex assembled at the cos site prior to DNA maturation and packaging. application/pdf en_US Copyright is held by the individual authors. bacteriophage lambda; cooperativity; integration host factor; protein-DNA interactions; sedimentation equilibrium; sedimentation velocity Biophysics Virology medicinal chemistry Cooperative Assembly of Terminase and Integration Host Factor at the Packaging Initiation Site of Bacteriophage Lambda Thesis Delay release for 2 years -- then make Open Access open.access