HKUST Institutional Repository >
BICH Doctoral Theses >
Please use this identifier to cite or link to this item:
|Title: ||General replicators and replication origins in human cells and the role of yeast ORC in mediating histone methylation|
|Other Titles: ||Role of yeast ORC in mediating histone methylation|
|Authors: ||Kan, Junsuo|
|Issue Date: ||2009 |
|Abstract: ||Initiation of DNA replication is controlled by the cis-acting replicators and the trans-acting initiator and initiation proteins that interact with replicators to initiate replication at replication origins. The replicators and origins have been defined as specific DNA sequences in yeasts. However, although over one hundred unique metazoan replicators/origins have been identified, searching for general metazoan replicators is less fruitful. Here, I report a group of general human replicators identified by chromatin immunoprecipitation (ChIP) using antibodies against ORC and MCM proteins followed by unbiased cloning of the precipitated DNA. The interactions of the putative replicators with multiple initiation proteins were confirmed by ChIP assays with multiple PCR primer pairs to detect the precipitated DNA and a more stringent re-ChIP assay. The putative replicators are part of LINE1 (L1; long interspersed nuclear elements), and are homologous to one another and also to tens of thousand other loci distributed relatively evenly in the human genome, with a density of one in every ~100 kb. The DNA sequence, per se, shares some features of replication origins. It was also demonstrated that the replication origins were located near the replicators using nascent strand abundance assays with either alkaline purified or BrdU labeled nascent DNA. In addition, the DNA combing assay confirmed that the in vivo DNA replication was initiated in the area of the putative replicators. Furthermore, episomal replication assays showed that the replicator/origin-containing plasmids, but not the control plasmids, became Dpn1-resisitant after transfection into human cells, indicating duplication of the replicator/origin-containing plasmids. This study identified a group of human replicators and replication origins that are part of L1, and they may represent the general replicators and replication origins responsible for the duplication of the bulk of the human genome.
The hetero-hexameric origin recognition complex (ORC) has been implicated in many cellular activities, including DNA replication, transcriptional control, heterochromatin assembly, centromere and telomere function, and so on. Here, I report a new function for ORC in mediating histone methylation. Using the yeast two-hybrid system I identify a physical interaction between Orc2p and Spp1p, a member of the Set1 complex, and I demonstrate the interaction between the endogenous ORC and Spp1p by co-immunoprecipitation (co-IP) from yeast extracts. Furthermore, I find that Orc2p physically interacts with trimethylated histone 3 lysine 4 (H3K4) on chromatin by co-IP. Finally, I show that the tri-methylation of H3K4 is decreased in orc2-1 cells and abolished in orc2-1 spp1⊿ double mutants. These data reveal a novel facet of ORC in mediating histone methylation in collaboration with Spp1p, and demonstrate a connection between ORC and chromatin structure via the Set1 complex.|
|Description: ||Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2009|
xviii, 126 p. : ill. (some col.) ; 30 cm
HKUST Call Number: Thesis BICH 2009 Kan
|Appears in Collections:||BICH Doctoral Theses|
Files in This Item:
All items in this Repository are protected by copyright, with all rights reserved.