Regulation Of Initiation Of DNA Replication In Budding Yeast

Most of our knowledge about initiation of eukaryotic DNA replication are derived from the study of budding yeast Saccharomyces cerevisiae. The study of DNA replication factors in budding yeast will help us get a deeper understanding of the mechanism of initiation of eukaryotic DNA replication, which will be greatly promoted by establishing in vitro replication system in budding yeast. Here, the first part of this thesis is a work about the establishing an in vitro replication system in budding yeast, and the second part is a work about DNA replication and cell cycle.In the first part, we attempted to employ the high efficiency of Xenopus egg extracts to establish an in vitro replication system in budding yeast. We first found out the preparation of intact budding yeast nuclei suitable for substrates for replication in Xenopus egg extracts. When intact G1-nuclei were introduced into Xenopus egg extracts, robust DNA synthesis was observed after a brief lag period. This DNA synthesis was aphidicolin-sensitive, indicating it was mediated by replicative DNA polymerase. Using the intact G1-nuclei prepared from an isogenic yeast strain lacking mitochondrial DNA (rho0), we demonstrated that the DNA synthesis observed within the first 10-20 min was largely due to mitochondrial DNA synthesis, whereas nuclear DNA synthesis did not begin until after a 10-20 min lag period. The aphidicolin-sensitive DNA synthesis resulted from semiconservative DNA replication as confirmed by neutral Cs2SO4 equilibrium gradient centrifugation. Alkaline Cs2SO4 equilibrium gradient centrifugation revealed that DNA synthesis was achieved by replication rather than DNA repair. A comparison of the properties of DNA synthesis within G1-phase and S-phase nuclei incubatedin Xenopus egg extract further confirmed that DNA replication stimulated by Xenopus egg cytosol resulted from de novo initiation of replication in the extract and not the extension of preprimed DNA templates. Nearly all G1-phase nuclei were stimulated to enter S-phase in vitro, and up to 40% of the total genomic DNA were replicated within 90 min. We conclude that Xenopus egg cytosol can efficiently initiate DNA replication within intact G1-phase yeast nuclei.Using neutral-neutral 2D gel electrophoresis analysis, we demon- strated that aphidicolin can accumulate replication mediates and thus improve the detection of replication intermediates generated in vitro. Replication intermediates were most easily detectable in samples supplemented with 3 (g/ml aphidicolin. Using this method, we demonstrated that Xenopus egg extract did not preferentially recognize the 2-(m replication origin, regardless of the integrity of the yeast nuclear envelope, and that initiation within intact yeast nuclei by Xenopus egg extract can take place at many sites that do not normally function as origins in living yeast cells.The second part of the thesis started from Mcm10 gene involved in DNA replication in budding yeast. mcm10-1 mutant is temperature-sensitive and its growth was arrested at 37℃. We got a mcm10-1 suppressor by a genetic screen, which was named as WJ-1. Viability assay indicated that WJ-1 cells were viable at 37℃ and were arrested, while longer incubation at 14℃ caused cells to die. The mcm10 gene in WJ-1 cells was not reversionally mutated and still maintained the allele in mcm10-1 cells. FACS analysis revealed that WJ-1 cells showed a normal cell cycle pattern at 37℃, and an abnormal one at 14℃. This abnormal cell cycle may be due to rereplication of genomic DNA, which was supported by the DAPI staining of nuclei.By screening yeast genomic DNA library, we found plasmids harboring Mcm2 gene essential for DNA replication can complement the cold-sensitivity of WJ-1 cells well. Overexpression of Mcm10 and Mcm2 genes suggested that mcm2 gene might be mutated in WJ-1 cells. Direct sequencing demonstrated that the mcm2 gene was mutated and the mutationwas a serine to tyrosine substitution at amino acid 619 that lies in MCM box of Mcm2 protein. Sequence analysis sh...