Biochemical analysis of proteins in the telomere maintenance pathway

Telomere;Telomerase;Chaperone;Cellular dynamics

Dezwaan, Diane C.

关键词: Telomere;    Telomerase;    Chaperone;    Cellular dynamics;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/18345/DeZwaan_Diane.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Eukaryotic linear chromosomes culminate in nucleoprotein structures designatedtelomeres. The terminal telomeric DNA consists of tandem repeats of a G-rich motif thatis established and maintained by the action of the specialized reverse transcriptase calledtelomerase. In addition to the function of telomerase, the telomere environment requiresan efficient means to assemble and disassemble a multitude of structures to operatecorrectly and to help achieve cellular homeostasis. Distinct protein assemblies arenucleated at telomeric DNA to both guard the ends from damage and lengthen the DNAafter replication. In yeast, Cdc13 recruits either Stn1-Ten1 to form a protective cap or thetelomerase holoenzyme to extend the DNA. I have established an in vitro yeast telomeresystem in which Stn1-Ten1-unextendable or telomerase-extendable states can beobserved. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between thetelomere capping and extending structures by modulating the DNA binding activity ofCdc13. The telomere length and telomerase telomere occupancy also appear to be yeastHsp90 dependent. Taken together, my data show that the Hsp82 chaperone facilitatestelomere DNA maintenance by promoting transitions between two operative complexesand by reducing the potential for binding events that would otherwise block the assemblyof downstream structures.The first telomerase cofactor identified was the budding yeast protein Est1, whichis conserved through humans. While it is evident that Est1 is required for telomere DNAmaintenance, understanding its mechanistic contributions to telomerase regulation hasbeen limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNAextension. Although Est1 displayed specific DNA and RNA binding, neither activityiiicontributed significantly to telomerase stimulation. Rather Est1 mediated telomeraseupregulation through direct contacts with the reverse transcriptase subunit. My studiesprovide insights into the molecular events used to control the enzymatic activity of thetelomerase holoenzyme.