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Date of Award

Summer 8-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The work discussed in this dissertation focuses on two distinct areas that are critical for genome maintenance. Both areas are focused on the regulation of the Saccharomyces cerevisiae checkpoint kinase Tel1 by the MRX complex. Tel1 kinase initiates cell cycle checkpoint in response to double-strand DNA breaks. Tel1 also plays a major role in telomere maintenance. Tel1’s function both in checkpoint signaling and telomere regulation is dependent on the Mre11-Rad50-Xrs2 complex. In Chapter II, I describe a robust biochemical approach aimed at reconstituting the initial stages of double-strand DNA break response using purified proteins in order to address how the MRX complex and DNA orchestrate to activate Tel1 kinase. Our results demonstrate that double-stranded DNA and MRX activate Tel1 synergistically. This work revealed a DNA length dependent stimulation of Tel1, with long, nucleosome-free duplex DNA being the preferred effector for full Tel1 activation. Our work also highlights there is no requirement for double-stranded DNA ends for Tel1 activation by MRX in vitro. Our data show Rad50 is the most critical subunit as no stimulation of Tel1 is seen with the Mre11-Xrs2 pair and DNA, which is in agreement with reported genetic observations. This stimulatory effect of Rad50 is absolutely dependent on its ATP binding activity. This work provides a comprehensive model into how individual subunits of MRX collaborate with DNA to activate Tel1 kinase. The second portion of this thesis discusses work employing the biochemical assay described in Chapter II to investigate the regulation of Tel1 kinase and MRX at telomeres. Similar to several other checkpoint and DNA repair factors, Tel1 and MRX are involved in telomere regulation. Unique architectural organization and remodeling of telomeric DNA by telomere binding proteins prevents telomeres from inappropriate recognition and repair as intrachromosomal DNA breaks. In budding yeast, Rif2 is one of the main proteins involved in checkpoint response suppression at long telomeres. MRX has been shown to recruit Tel1 specifically to short telomeres, resulting in the recruitment of telomerase and the elongation of short telomeres. Conversely, based on genetic observations Rif2 has been suggested to attenuate elongation of long telomeres through the MRX-Tel1 pathway. Our studies show Rif2 directly inhibits MRX-dependent activation of Tel1 kinase. The inhibitory role of Rif2 is mediated through its conserved N-terminal domain. Our data demonstrate that Rif2 exerts its inhibition by modulating the ATPase activity of Rad50. Investigation of an allosteric Rad50 ATPase mutant that maps outside of the conserved ATP binding domain suggest Rif2 discharges the ATP-bound form of Rad50, which is a state conducive for Tel1 activation. Taken together these data point to a novel role of Rif2 in regulating Tel1 kinase through the Rad50 subunit of MR

Language

English (en)

Chair and Committee

Peter Burgers

Committee Members

John Majors, Roberto Galletto, Eric Galburt, Alessandro Vindigni,

Comments

Permanent URL: https://doi.org/10.7936/k1h0-ab22

Available for download on Tuesday, August 15, 2119

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