{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/122523"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/122523","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Mechanism and importance of Mcm2-7 double-hexamer formation during DNA replication initiation","abstract":"All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head \"double hexamer\". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.","abstract_html":"All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head "double hexamer". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.","abstract_has_math":false,"creators":["Champasa, Kanokwan."],"institution":"Massachusetts Institute of Technology","degree_name":"Doctoral","degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Biology","school":null,"contributors":[],"advisors":["Stephen P. Bell."],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019","date_published":"2019","updated_at":"2026-07-22T22:22:01Z","subjects":["Biology."],"languages":["eng"],"rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/1721.1/122523","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Stephen P. Bell."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Biology","Bio"]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. Department of Biology."]},{"key":"dc:creator","label":"Author","values":["Champasa, Kanokwan."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2019-10-11T22:00:12Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2019-10-11T22:00:12Z"]},{"key":"dc:date.issued","label":"Date","values":["2019"]},{"key":"dc:publisher","label":"Institution","values":["Massachusetts Institute of Technology"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctoral"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology."]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/1721.1/122523"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019","Cataloged from PDF version of thesis.","Includes bibliographical references."]},{"key":"dc:description.abstract","label":"Abstract","values":["All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head \"double hexamer\". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.","The order of events during the first helicase loading has been established, but the mechanism of double-hexamer formation remains unclear. Because the two helicases interact at their N-terminal domains, these regions represent potential mediators of double-hexamer formation. This thesis outlines the potential mechanism and the importance of double-hexamer formation. A conserved motif within Mcm2-7 N-terminal region is required for stable double-hexamer formation and cell viability. Single-molecule analyses of Mcm2-7 containing a mutation within this motif indicated that this mutant form double-hexamer interactions briefly before the two hexamers come apart. Interestingly, after double-hexamer dissolution, the two mutant helicases do not form subsequent double-hexamer interaction. Both wild-type and the mutant Mcm2-7 exhibit double-hexamer interaction rapidly after the arrival of the second Mcm2-7.","Together, these data support the model that double-hexamer formation is coordinated with loading of the second Mcm2-7. Finally, the requirement of the double hexamer during helicase activation was investigated using Mcm2-7 complex containing the mutant that inhibits double-hexamer formation. The double hexamer is not essential for recruitment of three critical helicase-activation proteins, but it is required for initial origin DNA unwinding. These findings identify a crucial motif for stable double-hexamer formation and suggest that DNA unwinding is the first step in replication initiation that requires double-hexamer form of the helicases."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:title","label":"Title","values":["Mechanism and importance of Mcm2-7 double-hexamer formation during DNA replication initiation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Stephen P. Bell."],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Biology","Bio"],"dc:contributor.other":["Massachusetts Institute of Technology. Department of Biology."],"dc:creator":["Champasa, Kanokwan."],"dc:date.accessioned":["2019-10-11T22:00:12Z"],"dc:date.available":["2019-10-11T22:00:12Z"],"dc:date.issued":["2019"],"dc:description":["Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019","Cataloged from PDF version of thesis.","Includes bibliographical references."],"dc:description.abstract":["All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head \"double hexamer\". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.","The order of events during the first helicase loading has been established, but the mechanism of double-hexamer formation remains unclear. Because the two helicases interact at their N-terminal domains, these regions represent potential mediators of double-hexamer formation. This thesis outlines the potential mechanism and the importance of double-hexamer formation. A conserved motif within Mcm2-7 N-terminal region is required for stable double-hexamer formation and cell viability. Single-molecule analyses of Mcm2-7 containing a mutation within this motif indicated that this mutant form double-hexamer interactions briefly before the two hexamers come apart. Interestingly, after double-hexamer dissolution, the two mutant helicases do not form subsequent double-hexamer interaction. Both wild-type and the mutant Mcm2-7 exhibit double-hexamer interaction rapidly after the arrival of the second Mcm2-7.","Together, these data support the model that double-hexamer formation is coordinated with loading of the second Mcm2-7. Finally, the requirement of the double hexamer during helicase activation was investigated using Mcm2-7 complex containing the mutant that inhibits double-hexamer formation. The double hexamer is not essential for recruitment of three critical helicase-activation proteins, but it is required for initial origin DNA unwinding. These findings identify a crucial motif for stable double-hexamer formation and suggest that DNA unwinding is the first step in replication initiation that requires double-hexamer form of the helicases."],"dc:description.degree":["Ph. D."],"dc:identifier.uri":["https://hdl.handle.net/1721.1/122523"],"dc:language.iso":["eng"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"dc:rights.uri":["http://dspace.mit.edu/handle/1721.1/7582"],"dc:subject":["Biology."],"dc:title":["Mechanism and importance of Mcm2-7 double-hexamer formation during DNA replication initiation"],"dc:type":["Thesis"],"thesis:degree_name":["Doctoral"]},"updated_at":"2026-07-22T22:22:01Z"}