{"id":{"repo_id":"washington","oai_identifier":"oai:digital.lib.washington.edu:1773/24142"},"canonical_url":"https://search.dev.ndltd.org/etd/washington/oai:digital.lib.washington.edu:1773/24142","repository":{"repo_id":"washington","name":"University of Washington","base_url":"https://digital.lib.washington.edu/server/oai/request"},"display":{"title":"Cooperative Assembly of Terminase and Integration Host Factor at the Packaging Initiation Site of Bacteriophage Lambda","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Sanyal, Saurarshi Jyoti"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Catalano, Carlos E"],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013-11-14","date_published":"2013-11-14","updated_at":"2026-07-24T05:58:01Z","subjects":["bacteriophage lambda; cooperativity; integration host factor; protein-DNA interactions; sedimentation equilibrium; sedimentation velocity"],"languages":["en_US"],"rights":["Copyright is held by the individual authors."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1773/24142","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Catalano, Carlos E"]},{"key":"dc:creator","label":"Author","values":["Sanyal, Saurarshi Jyoti"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2013-11-14T20:53:35Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2015-12-14T17:55:48Z"]},{"key":"dc:date.issued","label":"Date","values":["2013-11-14"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["bacteriophage lambda; cooperativity; integration host factor; protein-DNA interactions; sedimentation equilibrium; sedimentation velocity"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright is held by the individual authors."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["Sanyal_washington_0250E_12247.pdf"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1773/24142"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis (Ph.D.)--University of Washington, 2013"]},{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Cooperative Assembly of Terminase and Integration Host Factor at the Packaging Initiation Site of Bacteriophage Lambda"]}]}],"canonical_facts":{"dc:contributor.advisor":["Catalano, Carlos E"],"dc:creator":["Sanyal, Saurarshi Jyoti"],"dc:date.accessioned":["2013-11-14T20:53:35Z"],"dc:date.available":["2015-12-14T17:55:48Z"],"dc:date.issued":["2013-11-14"],"dc:description":["Thesis (Ph.D.)--University of Washington, 2013"],"dc:description.abstract":["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."],"dc:format.mimetype":["application/pdf"],"dc:identifier.other":["Sanyal_washington_0250E_12247.pdf"],"dc:identifier.uri":["http://hdl.handle.net/1773/24142"],"dc:language.iso":["en_US"],"dc:rights":["Copyright is held by the individual authors."],"dc:subject":["bacteriophage lambda; cooperativity; integration host factor; protein-DNA interactions; sedimentation equilibrium; sedimentation velocity"],"dc:title":["Cooperative Assembly of Terminase and Integration Host Factor at the Packaging Initiation Site of Bacteriophage Lambda"],"dc:type":["Thesis"]},"updated_at":"2026-07-24T05:58:01Z"}