{"id":{"repo_id":"texas","oai_identifier":"oai:repositories.lib.utexas.edu:2152/82472"},"canonical_url":"https://search.dev.ndltd.org/etd/texas/oai:repositories.lib.utexas.edu:2152/82472","repository":{"repo_id":"texas","name":"University of Texas","base_url":"https://repositories.lib.utexas.edu/server/oai/request"},"display":{"title":"Single-molecule studies reveal mechanisms of human DNA double-strand break repair","abstract":"DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation.","abstract_html":"DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation.","abstract_has_math":false,"creators":["Myler, Logan Ross"],"institution":"The University of Texas at Austin","degree_name":"Doctor of Philosophy","degree_level":"Doctoral","degree_discipline":"Cell and molecular biology","degree_department":null,"school":null,"contributors":[],"advisors":["Paull, Tanya T.","Finkelstein, Ilya J."],"committee_chairs":[],"committee_members":["Matouschek, Andreas T","Miller, Kyle M","Liu, Hung-wen"],"year":2018,"date_issued":"2018-06-13","date_published":"2018-06-13","updated_at":"2026-07-24T05:00:58Z","subjects":["DNA","Double-strand break","MRN","Exo1","Resection","Resectosome","Homologous recombination"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://dx.doi.org/10.26153/tsw/9477"],"render_values":[{"text":"http://dx.doi.org/10.26153/tsw/9477","href":"http://dx.doi.org/10.26153/tsw/9477","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152/82472","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Paull, Tanya T.","Finkelstein, Ilya J."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Matouschek, Andreas T","Miller, Kyle M","Liu, Hung-wen"]},{"key":"dc:creator","label":"Author","values":["Myler, Logan Ross"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2020-08-06T15:04:59Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2020-08-06T15:04:59Z"]},{"key":"dc:date.issued","label":"Date","values":["2018-06-13"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Cell and molecular biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["The University of Texas at Austin"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["DNA","Double-strand break","MRN","Exo1","Resection","Resectosome","Homologous recombination"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/2152/82472","http://dx.doi.org/10.26153/tsw/9477"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Single-molecule studies reveal mechanisms of human DNA double-strand break repair"]}]}],"canonical_facts":{"dc:contributor.advisor":["Paull, Tanya T.","Finkelstein, Ilya J."],"dc:contributor.committeemember":["Matouschek, Andreas T","Miller, Kyle M","Liu, Hung-wen"],"dc:creator":["Myler, Logan Ross"],"dc:date.accessioned":["2020-08-06T15:04:59Z"],"dc:date.available":["2020-08-06T15:04:59Z"],"dc:date.issued":["2018-06-13"],"dc:description.abstract":["DNA damage is ubiquitous to all organisms and very complex pathways have evolved to recognize and repair these lesions. The most deleterious DNA damages are double-strand breaks (DSBs), and a single unrepaired DSB can lead to cell death. In human cells, there exist two canonical pathways of DSB repair: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Two protein complexes that rapidly bind DNA ends coordinate these separate pathways: the Ku70-Ku80 heterodimer (Ku) and the Mre11-Rad50-Nbs1 complex (MRN), respectively. Ku encircles the DNA ends and recruits other factors, such the kinase DNA-PKcs, to bluntly ligate the ends back together. In contrast, MRN along with the long-range nuclease Exo1 and helicase BLM digests the DNA to create long 3’ single-stranded DNA overhangs, which are rapidly bound by the single-stranded DNA binding protein RPA. Next, Rad51 replaces RPA and facilitates strand exchange into a homologous chromosome to resynthesize the missing information in a largely error-free way. Despite the importance of DSB repair, many of the underlying mechanisms by which these molecular machines dynamically assemble and carry out the repair process have remained unknown. Here, I use a combination of ensemble biochemical assays as well as high-throughput single-molecule microscopy to visualize the repair process. I have observed two main steps of the repair process: initiation of HR by MRN and long-range resection by Exo1. I have found that MRN locates DSBs by a sliding mechanism that allows it to load on Ku-blocked ends. Then, once it reaches the end, MRN removes DNA-PK and recruits Exo1 and BLM in order to promote long-range digestion of the DNA. Finally, the Exo1/BLM resectosome is attenuated by phosphorylation of RPA. Overall, I have characterized the initiation and regulation of DSBR. This will lead to a new understanding of the ways in which these deleterious lesions are repaired and will contribute to understanding cancer as well as techniques for genetic manipulation."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://hdl.handle.net/2152/82472","http://dx.doi.org/10.26153/tsw/9477"],"dc:language.iso":["en"],"dc:subject":["DNA","Double-strand break","MRN","Exo1","Resection","Resectosome","Homologous recombination"],"dc:title":["Single-molecule studies reveal mechanisms of human DNA double-strand break repair"],"dc:type":["Thesis"],"thesis:degree_discipline":["Cell and molecular biology"],"thesis:degree_level":["Doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["The University of Texas at Austin"]},"updated_at":"2026-07-24T05:00:58Z"}