Studies of ORC dimerization and de-dimerization in the budding yeast saccharomyces cerevisiae

DNA replication is fundamental to life and for organisms to transmit their genetic information to the next generation. A crucial step in DNA replication is replication initiation [1]. This topic has been extensively studied in eukaryotes from yeast to mammalian cells, including investigation in replication initiation proteins, mechanisms controlling replication initiation, and complexes executing the function of initiating replication [2]. Despite all the understanding we have gained in this field, how does an ORC single hexamer as an asymmetric platform recruit the symmetric pre-replicative complex is still a major problem for research trying to construct the replication licensing model. Previously our lab has found evidence that ORC dimerizes, i.e., forming double hexamers. In this thesis, we performed several experiments to further study ORC dimerization and de-dimerization, as well as the potential mechanisms controlling these processes. The first experiment was to use the yeast two-hybrid assay to map the regions which are crucial for ORC dimerization. The gene expressing Orc1p and Orc6p, which are known to self-interact from the previous work of our lab, were dissected, and a series of plasmids were constructed and used to transform the yeast strains to carry out yeast two-hybrid analysis. We found out that a.a. 246-463 of Orc1p and a.a.224-333 of Orc6p have interaction with Orc1p and Orc6p, respectively, and also with the fragments themselves. The second experiment was chromatin binding essay preformed with wild type yeast cells. Based on the previous results from our lab that ORC dimerization could only occur in G1 but not S or G2/M phase, and that newly synthesized ORC could bind to chromatin only during the M-to-G1 transition, we compared the signal ratios of Orc3p/Histone H3 and Mcm2p/Histone H3 on chromatin in different time points when yeast cells were synchronized in M phase and then released into G1 phase. We found that the signal of Orc3p on chromatin began at 10-20 min after release from M phase and doubled at 50 min after release, while Mcm2p chromatin loading was delayed, starting at 20-30 min. These results indicate that ORC dimerization occurs before MCM loading onto chromatin, further supporting the conclusion from our lab that ORC dimerization is required for MCM chromatin loading. We also performed co-immunoprecipitation (co-IP) assay to examine the time course of ORC dimerization. A plasmid co-expressing Orc4-Myc and Orc4-FLAG was used to transform yeast, and cell samples were harvested at different time points during the M-to-G1 and G1-to-S transitions and subjected to co-IP assay. Using anti-FLAG and anti-Myc antibodies for co-IP followed immunobloting, we found out that the co-IP signal appeared at around 20 min after M release. Similarly, we found out that the signal of Orc1-Myc and Orc1-FLAG appeared at around 20 min after M release, and disappeared at 40 min after G1 release. From the data of chromatin binding assay with WT cells, we found out that the phosphorylation status of Orc6p is synchronous to ORC dimerization and de-dimerization, with de-phosphorylation associating with dimerization. To investigate if the phosphorylation status of Orc6p and Orc2p controls ORC dimerization, we transformed wild type yeast cells with pESC-FLAG-Orc6/Orc6-Myc, pESC-FLAG-Orc1/Orc1-Myc or pESC-FLAG-Orc3/Orc3-Myc into orc2A, orc6A mutant cells in which Orc2 and Orc6 cannot be phosphorylated. We then performed chromatin binding assay on the mutant cells released from G1 phase. We found that ORC could form dimers in S and M phase in the mutant cells, suggesting that Orc2p/Orc6p de-phosphorylation is necessary for ORC dimerization.