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JagatBudhathoki_PhD Dissertation.pdf (24.75 MB)

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Interactions of RecQ-Family Helicases with G-quadruplex Structures at the Single Molecule Level

Budhathoki, Jagat B
http://rave.ohiolink.edu/etdc/view?acc_num=kent1467765011

Abstract Details

2016, PHD, Kent State University, College of Arts and Sciences / Department of Physics.
Human RecQ helicases are members of superfamily 2 helicases and consist of five proteins: RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. RecQ helicases take part in various aspects of DNA metabolism including homologous recombination, replication, telomere maintenance, and DNA damage signaling. Mutations in BLM, WRN, and RECQ4 are associated with Bloom, Werner, and Rothmund-Thomson syndromes, respectively, which are various forms of genetic disorders. A significant cellular function of RecQ helicases is their interaction with diverse DNA structures including non-canonical structures such as guanine-quadruplex (GQ) and Holliday junction. G-quadruplex structures (GQs) can potentially form in guanine-rich segments of the genome, particularly during DNA replication or repair when double stranded DNA (dsDNA) is unwound. The formation and modulation of GQs in live human cells have recently been demonstrated via direct imaging and various indirect measurements. Interestingly, potentially GQ forming sequences are particularly more frequent in promoter regions of certain oncogenes. When formed in promoters, GQs are demonstrated to regulate transcription level gene expression. GQs are typically thermally more stable compared to dsDNA, and they pose a threat to genome integrity unless they are unfolded. Due to their ability to interact with a variety of DNA substrates, GQs are a natural target for RecQ helicases. Therefore, we studied the interaction of GQs with BLM, WRN, and RECQ5 using single molecule Förster Resonance Energy Transfer (smFRET) and bulk biophysical methods. Our studies on BLM and WRN provided the first evidence of unfolding of GQ without the requirement of ATP, when these helicases bind in the vicinity of GQ. This is similar to how Replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein, destabilizes GQ by binding to adjacent ssDNA overhang. We found that BLM-mediated GQ unfolding is most efficient in the ATPγS-bound state and least efficient in the ADP-bound state, which correlates with higher stability of BLM binding to ssDNA in the presence of ATPγS compared to ADP. In the presence of ATP, a much higher rate of BLM-mediated GQ unfolding was observed compared to its absence. We also found that the rate of GQ unfolding significantly increased with increasing BLM concentration, which suggests low processivity for BLM-mediated GQ unfolding activity. Our studies on RECQ5 showed weak GQ destabilization only in the presence of ATP. The rate of GQ unfolding for RECQ5 compared with that of BLM for the same DNA construct was found to be an order of magnitude smaller. Based on these, we conclude that RECQ5 is a weak GQ destabilizer and is probably not primarily involved in destabilizing GQs, but it may assist more robust helicases such as BLM and WRN in this task. Despite these differences in their GQ unfolding activity, we observed that both BLM and RECQ5 show a similar reeling activity, i.e. proteins localize in the vicinity of dsDNA/ssDNA junction and pull the downstream ssDNA overhang. We also verified that reeling is not essential for GQ unfolding. While BLM unfolded a downstream GQ after reeling an ssDNA overhang, RECQ5 was not able to do so. These studies provide a number of important insights on the similarities and differences between RecQ family helicases, and have implications for their cellular function and for the potential role of GQ in aforementioned syndromes associated with these helicases.
Hamza Balci, Dr. (Advisor)
John Portman, Dr. (Committee Member)
Elizabeth Mann, Dr. (Committee Member)
Qi-Huo Wei, Dr. (Committee Member)
Gail C. Fraizer, Dr. (Committee Member)
Edgar Kooijman, Dr. (Other)
272 p.
Biomechanics; Biophysics
RecQ helicases, BLM, WRN, RECQ5, G-quadruplex, smFRET, three-color FRET, GQ unfolding, ssDNA reeling, ATP

Recommended Citations

Citations

  • Budhathoki, J. B. (2016). Interactions of RecQ-Family Helicases with G-quadruplex Structures at the Single Molecule Level [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1467765011

    APA Style (7th edition)

  • Budhathoki, Jagat. Interactions of RecQ-Family Helicases with G-quadruplex Structures at the Single Molecule Level. 2016. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1467765011.

    MLA Style (8th edition)

  • Budhathoki, Jagat. "Interactions of RecQ-Family Helicases with G-quadruplex Structures at the Single Molecule Level." Doctoral dissertation, Kent State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1467765011

    Chicago Manual of Style (17th edition)

kent1467765011
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© 2016, all rights reserved.
This open access ETD is published by Kent State University and OhioLINK.