| dc.identifier.uri | http://hdl.handle.net/11401/78380 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.format.mimetype | Application/PDF | en_US |
| dc.language.iso | en_US | |
| dc.type | Thesis | |
| dcterms.abstract | DNA replication in eukaryotes is a highly dynamic process. A fully-assembled eukaryotic replisome is a multiprotein machine that contains over two dozen proteins, including DNA polymerases, sliding clamps, replicative helicase, and primase along with several factors that participate in cell cycle and checkpoint control. But the formation of the replisome is a lengthy, intricate, and highly regulated process. By taking advantage of the recent advent of the direct electron detector and powerful image analysis algorithm, I have worked with my colleagues and collaborators to solve the cryo-EM structures of four intermediate complexes in different stages of replisome assembly. The first structure is an ORC-Cdc6-Cdt1-Mcm2-7(OCCM) helicase-loading intermediate, which revealed the loading mechanism of the first Cdt1-bound Mcm2-7 hexamer onto DNA by the ORC-Cdc6. The second structure of Mcm2-7 double hexamer(DH) loaded and assembled on dsDNA suggested a potential mechanism for the DNA strand separation and lagging-strand extrusion. The third structure of the Cdc45/Mcm2-7/GINS active helicase (CMG) revealed two distinct conformations that led us to propose a pumpjack-like mechanism of helicase translocation on DNA. The forth structure of CMG bound to a doubly-blocked forked DNA in the presence of ATP suggested a new N-tier to C-tier polarity of CMG translocation on DNA. Taken together, our single-particle cryo-EM studies have established the overall architecture of the eukaryotic replisome. Contrary to the long-held view that the polymerases and primase trail behind the helicase, we found the leading strand polymerase epsilon trails behind, but the lagging strand primase-polymerase alpha rides ahead of the helicase, and importantly, the C-tier ATPase motor ring of the helicase pushes from behind with the N-tier ring in front separating the DNA duplex. Our work has provided important insights into eukaryotic DNA replisome assembly and function. | |
| dcterms.available | 2018-07-10T19:14:30Z | |
| dcterms.contributor | Garcia-Diaz, Miguel | en_US |
| dcterms.contributor | Li, Huilin | en_US |
| dcterms.contributor | Glynn, Steven | en_US |
| dcterms.contributor | Thanassi, David | en_US |
| dcterms.creator | Yuan, Zuanning | |
| dcterms.dateAccepted | 2018-07-10T19:14:30Z | |
| dcterms.dateSubmitted | 2018-07-10T19:14:30Z | |
| dcterms.extent | pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.identifier | http://hdl.handle.net/11401/78380 | |
| dcterms.issued | 2017-01-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Submitted by Jason Torre (fjason.torre@stonybrook.edu) on 2018-07-10T19:14:29Z
No. of bitstreams: 1
Yuan_grad.sunysb_0771E_13585.pdf: 4483844 bytes, checksum: 399f31f4c5a69cf08edfab4c82b380c4 (MD5) | en |
| dcterms.provenance | Made available in DSpace on 2018-07-10T19:14:30Z (GMT). No. of bitstreams: 1
Yuan_grad.sunysb_0771E_13585.pdf: 4483844 bytes, checksum: 399f31f4c5a69cf08edfab4c82b380c4 (MD5)
Previous issue date: 2017-01-01 | en |
| dcterms.subject | cryo-electron microscopy | |
| dcterms.title | Structural insights into the assembly and function of the eukaryotic DNA replisome | |
| dcterms.type | Thesis | |
