<p>TRF1 is an essential shelterin protein that binds to double stranded telomeric DNA. TRF1 is best known for its role as a negative regulator of telomere length. Post-translational modifications, like phosphorylation, have been shown to regulate TRF1 function in cells. Mass spectrometric analysis revealed three potential TRF1 phosphorylation sites, threonine 271, serine 279 and threonine 305 <em>in vivo</em>. To analyze the function of these three potential phosphorylation sites, phosphomimic (aspartic acid, D) and non-phosphorylatable (alanine, A) mutations were made to be analyzed <em>in vitro</em>. Through <em>in vitro</em> gel shift assays, the phosphomimic mutation at threonine 271 mutant exhibits a DNA binding defect, whereas serine 279 and threonine 305 mutants have no binding defects. However, <em>in vivo</em> analysis needs to be conducted in order to determine whether this binding defect is authentic.</p> <p>PIN2 is a TRF1 isoform, which is identical in its protein sequence with the exception of 20 amino acids (residue 296-316 of TRF1). The second study looks to characterize any potential functional differences between PIN2 and TRF1. <em>In vitro</em>, CDK1 kinase assay was conducted on TRF1 and PIN2 mutants to assess whether there was a difference in phosphorylation. The kinase assay revealed that both PIN2 threonine 351 and TRF1 threonine 371 are both phosphorylated by CDK1. However, the net phosphorylation level of PIN2 by CDK1 is far lower than the net phosphorylation level of TRF1. An <em>in vitro</em> gel shift assay was also conducted to analyze the binding difference between TRF1 threonine 371 and PIN2 threonine 351 mutants. The DNA binding assay revealed that TRF1 threonine 371D mutant has a binding defect, whereas PIN2 threonine 351D mutant has no binding defect. However, this data needs to be verified to determine whether a PIN2 threonine 351D mutant behaves like a phosphomimic.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/15247 |
| Date | 26 September 2014 |
| Creators | Jeyanthan, Kajaparan |
| Contributors | Zhu, Xu-Dong, Junop, Murray, Biology |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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