Accurate and complete DNA replication is essential for maintaining the integrity of the genome. In eukaryotes, this process requires the coordinated action of numerous molecular machines. Based on yeast and animal model systems, we defined a set of fifty-one ?core DNA replication proteins? that are integral to the initiation, DNA synthesis, and Okazaki fragment maturation functions of DNA replication. We used computational analyses to identify putative homologs in the genomes of two plants, Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), providing the first comprehensive view of the core DNA replication machinery in plants. Our results indicated that the overall composition of this apparatus is conserved, but plants are unique in that multiple DNA replication genes exist as small gene families. Fourteen of the genes we annotated in this study have not been previously reported in the literature, and we have provided revised gene models for seventeen plant proteins. To better understand how the DNA replication machinery functions in plants, we cloned multiple subunits of the pre-replication complex (pre-RC) from Arabidopsis and generated antibodies against four key components of this complex ? AtORC1, AtORC2, AtMCM5, and AtMCM7. We demonstrated that the pre-RC is developmentally regulated in Arabidopsis and, consistent with a role in DNA replication, is abundant in proliferating tissues. We used immunocytochemical and biochemical methods to characterize MCM7 in plants. We observed two distinct localization patterns for plant MCM7 proteins. In most cells, MCM7 was nuclear and colocalized with DNA. In a small fraction of cells, MCM7 was dispersed throughout the cytoplasmic compartment. Biochemical analysis confirmed that MCM7 binds to chromatin and that it is present in the nucleus at least during the G1, S and G2 cell cycle stages. Together, these analyses support a model where the MCM complex is loaded onto DNA in late M and early G1, released into the nucleoplasm during S phase followed by a brief dispersion into the cytoplasmic compartment concurrent with nuclear envelope breakdown in mitosis.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-03132007-124000 |
| Date | 04 April 2007 |
| Creators | Shultz, Randall William |
| Contributors | Rebecca Boston, Jeffrey Thorne, William Thompson, George Allen, Steven Spiker |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-03132007-124000/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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