| The events of cell cycle take place in the order of timing and space, which ensures that cell division and chromosome segregation are completed with high fidelity. In principle, the ordering of cell cycle events could be accomplished by requiring the completion of previous event. Checkpoints respond to DNA damage and DNA replication block by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. Until the damage was repaired, the cell processed into division cycle. Checkpoint loss results in genomic instability and has been implicated in the evolution of normal cells into cancer cells. Some mammalian genes are widely studied such as P53 that controls the DNA damage checkpoint. The regulatory control is too confused to study in mammalian cells, the budding yeast is used as the primary example. There are only several groups who study checkpoint regulation with yeast in USA. They founded a S phase checkpoint regulation signal pathway. In this pathway, po12 gene senses DNA damage and replication blocks, and it transduces signals to Mecl, which activates Rad53. Rad53p arrest the cell cycle. WEB2 (wants El A badly2)gene has a sequence of 4500bp which is identical to Dna2. The locus was mapped to the right arm of chromosome 8. The protein encoded by WEB2 is 17. 5Kdal protein which has a 3'to 5'helicase activity. We suppose WEB2 has activities other than helicase. In this studies, a new defect checkpoint yeast WEB2 mutant is used to determine if it has a role in S phase checkpoint regulation. We also want to know the location and effect that WEB2 has on othergenes in checkpoint signal transduction pathways.Part1: A role of WEB2 gene in S phase checkpoint regulation of Sac-charomyces cerevisiaeWe used a WEB2 mutant which depends El A in plasmid for survival. But the El A is not replacing the function of WEB2 . With hydroxyurea( HU) , which blocks DNA replication, we could find if the mutant had defect in S phase checkpoint ,and conclude if WEB2 has a role in the signal transduction pathway.When WEB2 mutant and wild - type strain are treated with a factor for 3. 5 hours, the cells were synchronized in Gl phase, for that a factor can induce the expression of Farl protein , which inhibited CDK activity. Then the cells are released into YPG media containing 200mM HU. The cells process into S phase and , the wild - type cells are arrested in S phase by HU , but WEB2 mutant couldnt arrested in S phase by HU. FAGS analysis showed WEB2 mutant a less than 2C DNA content as wild - type. A significant percentage of WEB2 cells showed elongated spindles 3hr after release in HU, which is consistent with mi-totic entry. Wild - type cells treated with HU were large - budded with short spindles. WEB2 mutant showed inappropriate entry into mitosis with unreplicat-ed DNA indictive of S phase checkpoint deficiency. Survival rate curve indicated WEB2 mutant died rapidly in presence of 200mM HU. These data demonstrate that S checkpoint in WEB2 mutant is defective. We concluded WEB2 gene has a role in S checkpoint regulation , and it locates in the signal pathway founded by Elledge. WEB2 p is high homologous to human Dna2 helicase like protein, so the study would shed light on checkpoint regulation in eukaryote cells and the mechanism of cancer currence.Part2: RNR3 gene expression is regulated by WEB2 gene in S checkpoint signal pathway in S. cerevisiae-the location of WEB2 gene hi S checkpoint signalpathway of S. cerevisiae(1)Previous works suggest the role of WEB2 in S checkpoint regulation, wewant to make sure the location of WEB2 in signal transduction pathway. In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair. RNR3 encodes a large subunit of Ribonucleotide reductase ( RNR) in Saccharomyces cerevisiae, which catalyzes the rate - limiting step for deoxyribonucleotide production required for DNA synthesis.In this studies,...
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