A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding
Licensing of eukaryotic origins of replication requires DNA loading of two copies of the McM2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications, Ltd
2020
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| Online Access: | https://hdl.handle.net/1721.1/126039 |
| _version_ | 1811096688896507904 |
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| author | Champasa, Kanokwan Blank, Caitlin Friedman, Larry J Gelles, Jeff Bell, Stephen P |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Champasa, Kanokwan Blank, Caitlin Friedman, Larry J Gelles, Jeff Bell, Stephen P |
| author_sort | Champasa, Kanokwan |
| collection | MIT |
| description | Licensing of eukaryotic origins of replication requires DNA loading of two copies of the McM2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential McM4 motif that permit loading of two McM2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant McM2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant McM2-7 reveal a limited time window for double-hexamer formation following second McM2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, McM10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation. |
| first_indexed | 2024-09-23T16:47:28Z |
| format | Article |
| id | mit-1721.1/126039 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T16:47:28Z |
| publishDate | 2020 |
| publisher | eLife Sciences Publications, Ltd |
| record_format | dspace |
| spelling | mit-1721.1/1260392022-09-29T21:31:29Z A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding Champasa, Kanokwan Blank, Caitlin Friedman, Larry J Gelles, Jeff Bell, Stephen P Massachusetts Institute of Technology. Department of Biology Licensing of eukaryotic origins of replication requires DNA loading of two copies of the McM2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential McM4 motif that permit loading of two McM2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant McM2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant McM2-7 reveal a limited time window for double-hexamer formation following second McM2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, McM10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation. National Institute of General Medical Sciences (Grant GM52339) National Cancer Institute (Grant P30-CA14051) 2020-07-01T14:03:47Z 2020-07-01T14:03:47Z 2019-08 2019-01 2019-11-26T19:20:19Z Article http://purl.org/eprint/type/JournalArticle 2050-084X https://hdl.handle.net/1721.1/126039 Champasa, Kanokwan et al. "A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding." eLife 8: e45538 © 2019 Champasa et al. en http://dx.doi.org/10.7554/elife.45538 eLife Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf eLife Sciences Publications, Ltd eLife |
| spellingShingle | Champasa, Kanokwan Blank, Caitlin Friedman, Larry J Gelles, Jeff Bell, Stephen P A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title | A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title_full | A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title_fullStr | A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title_full_unstemmed | A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title_short | A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding |
| title_sort | conserved mcm4 motif is required for mcm2 7 double hexamer formation and origin dna unwinding |
| url | https://hdl.handle.net/1721.1/126039 |
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