{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/97690"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/97690","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Single-molecule studies of mechanical and helicase-catalyzed disruption of nucleic acid duplexes","abstract":"Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair.","abstract_html":"Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (&lt;15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair.","abstract_has_math":false,"creators":["Whitley, Kevin D"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Computnl Biology","degree_department":null,"school":null,"contributors":["Chemla, Yann R.","Aksimentiev, Oleksii","Selvin, Paul R.","Ha, Taekjip"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-08-10T20:32:51Z","date_published":"2017-08-10T20:32:51Z","updated_at":"2026-07-22T22:24:34Z","subjects":["Nucleic acids","Optical tweezers","Single-molecule","Fluorescence","Helicases"],"languages":["en"],"rights":["Copyright 2017 Kevin Whitley"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/97690","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Chemla, Yann R.","Aksimentiev, Oleksii","Selvin, Paul R.","Ha, Taekjip"]},{"key":"dc:creator","label":"Author","values":["Whitley, Kevin D"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2017-08-10T20:32:51Z","2019-08-11T09:15:09Z","2017-04-20","2017-05"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Computnl Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Nucleic acids","Optical tweezers","Single-molecule","Fluorescence","Helicases"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2017 Kevin Whitley"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/97690"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2019-05-01","The student, Kevin Whitley, accepted the attached license on 2017-04-12 at 11:09.","The student, Kevin Whitley, submitted this Dissertation for approval on 2017-04-12 at 11:25.","This Dissertation was approved for publication on 2017-04-20 at 13:55.","DSpace SAF Submission Ingestion Package generated from Vireo submission #10718 on 2017-08-10 at 15:05:26","Made available in DSpace on 2017-08-10T20:32:51Z (GMT). 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The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2019-05-01","The student, Kevin Whitley, accepted the attached license on 2017-04-12 at 11:09.","The student, Kevin Whitley, submitted this Dissertation for approval on 2017-04-12 at 11:25.","This Dissertation was approved for publication on 2017-04-20 at 13:55.","DSpace SAF Submission Ingestion Package generated from Vireo submission #10718 on 2017-08-10 at 15:05:26","Made available in DSpace on 2017-08-10T20:32:51Z (GMT). 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