| dc.identifier.uri | http://hdl.handle.net/11401/76947 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | The exchange of reciprocal information between homologous chromosomes in conjunction with sister chromatid cohesion ensures proper segregation of homologous chromosomes during Meiosis I. Improper segregation of homologous chromosomes during meiosis leads to chromosomal imbalance, which in humans can lead to genetic disorders such as Down syndrome (Trisomy 21) and Turner syndrome (monosomy of the X chromosome). Meiotic recombination begins with the initiation of double-strand breaks (DSBs) and the biased repair of these breaks by the homologous chromosome. This inter-homolog (IH) bias is upheld by the meiotic recombination checkpoint kinase Mek1. Mek1 is a meiosis specific serine/threonine kinase that prevents cells from progressing through meiosis I with unrepaired DSBs and also places a bias for meiotic DSBs to be repaired using the homologous chromosome. Although Mek1 has been previously shown to be necessary for proper chromosome segregation via its promotion of IH bias and checkpoint functions, non-phosphorylatable mutants of Mek1 substrates, with the exception of Mek1 itself, do not pheno-copy a mek1∆. This suggests that there are other Mek1 substrates that promote IH bias as well as the meiotic recombination checkpoint. To identify novel substrates of Mek1, a method for sporulating yeast out of synthetic medium was developed. This method allows the application of quantitative stable isotope labeling by amino acids in cell culture (SILAC) phosphoproteomics to meiosis, thereby allowing identification of novel Mek1 substrates in a global and unbiased manner. An analog sensitive allele of MEK1 (mek1-as) was used to conditionally inactivate Mek1 in dmc1∆-arrested cells containing heavy isotopes of arginine and lysine. Mek1 phosphosites are underrepresented in the heavy culture, resulting in light/heavy (L/H) phosphopeptide ratios ≥2. Known substrates of Mek1, including Rad54 and Mek1 itself, were identified as proof of principle. Phosphopeptides from the dmc1∆ mek1-as experiments were divided into three classes based on their L/H ratio. Motif analysis of the different classes revealed that the Mek1 consensus motif, RXXT, was specifically enriched in the Mek1 active culture. Thus, 16 RXXT proteins were identified as candidate Mek1 substrates, with Spp1 and Rad17 having the most functional relevance to the known functions of Mek1. Motif analysis of the phosphorylation events that become enriched in the Mek1 inactivated culture revealed a D/EXS/Tψ motif that matches the known consensus motif of Polo-Like Kinase or Cdc5 in budding yeast, leading to the identification of 10 candidate Cdc5 substrates. Finally, SILAC experiments using mek1-as and ntd80-arrested cells indicated that phosphorylation of the synaptonemal complex protein Zip1 protein is dependent upon Mek1. My main contribution to this collaboration with Dr. Xiangyu Chen were identifying the Zip1 phosphorylation sites characterized in (Chen et al., 2015). | |
| dcterms.abstract | The exchange of reciprocal information between homologous chromosomes in conjunction with sister chromatid cohesion ensures proper segregation of homologous chromosomes during Meiosis I. Improper segregation of homologous chromosomes during meiosis leads to chromosomal imbalance, which in humans can lead to genetic disorders such as Down syndrome (Trisomy 21) and Turner syndrome (monosomy of the X chromosome). Meiotic recombination begins with the initiation of double-strand breaks (DSBs) and the biased repair of these breaks by the homologous chromosome. This inter-homolog (IH) bias is upheld by the meiotic recombination checkpoint kinase Mek1. Mek1 is a meiosis specific serine/threonine kinase that prevents cells from progressing through meiosis I with unrepaired DSBs and also places a bias for meiotic DSBs to be repaired using the homologous chromosome. Although Mek1 has been previously shown to be necessary for proper chromosome segregation via its promotion of IH bias and checkpoint functions, non-phosphorylatable mutants of Mek1 substrates, with the exception of Mek1 itself, do not pheno-copy a mek1∆. This suggests that there are other Mek1 substrates that promote IH bias as well as the meiotic recombination checkpoint. To identify novel substrates of Mek1, a method for sporulating yeast out of synthetic medium was developed. This method allows the application of quantitative stable isotope labeling by amino acids in cell culture (SILAC) phosphoproteomics to meiosis, thereby allowing identification of novel Mek1 substrates in a global and unbiased manner. An analog sensitive allele of MEK1 (mek1-as) was used to conditionally inactivate Mek1 in dmc1∆-arrested cells containing heavy isotopes of arginine and lysine. Mek1 phosphosites are underrepresented in the heavy culture, resulting in light/heavy (L/H) phosphopeptide ratios ≥2. Known substrates of Mek1, including Rad54 and Mek1 itself, were identified as proof of principle. Phosphopeptides from the dmc1∆ mek1-as experiments were divided into three classes based on their L/H ratio. Motif analysis of the different classes revealed that the Mek1 consensus motif, RXXT, was specifically enriched in the Mek1 active culture. Thus, 16 RXXT proteins were identified as candidate Mek1 substrates, with Spp1 and Rad17 having the most functional relevance to the known functions of Mek1. Motif analysis of the phosphorylation events that become enriched in the Mek1 inactivated culture revealed a D/EXS/Tψ motif that matches the known consensus motif of Polo-Like Kinase or Cdc5 in budding yeast, leading to the identification of 10 candidate Cdc5 substrates. Finally, SILAC experiments using mek1-as and ntd80-arrested cells indicated that phosphorylation of the synaptonemal complex protein Zip1 protein is dependent upon Mek1. My main contribution to this collaboration with Dr. Xiangyu Chen were identifying the Zip1 phosphorylation sites characterized in (Chen et al., 2015). | |
| dcterms.available | 2017-09-20T16:51:30Z | |
| dcterms.contributor | Hollingsworth, Nancy M | en_US |
| dcterms.contributor | Neiman, Aaron | en_US |
| dcterms.contributor | Luk, Ed | en_US |
| dcterms.contributor | Futcher, Bruce. | en_US |
| dcterms.creator | Suhandynata, Raymond Theodore | |
| dcterms.dateAccepted | 2017-09-20T16:51:30Z | |
| dcterms.dateSubmitted | 2017-09-20T16:51:30Z | |
| dcterms.description | Department of Biochemistry and Structural Biology | en_US |
| dcterms.extent | 121 pg. | en_US |
| dcterms.format | Application/PDF | en_US |
| dcterms.format | Monograph | |
| dcterms.identifier | http://hdl.handle.net/11401/76947 | |
| dcterms.issued | 2016-12-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:30Z (GMT). No. of bitstreams: 1
Suhandynata_grad.sunysb_0771E_12939.pdf: 6691203 bytes, checksum: 381a13fb5115ac0130689c2a8d617b16 (MD5)
Previous issue date: 1 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Biochemistry -- Biology | |
| dcterms.title | Using Quantitative SILAC Proteomics to Identify Mek1 Substrates During Yeast Meiosis | |
| dcterms.type | Dissertation | |
