Physical and genetic interactions involving the nonhomologous end-joining factor XLF/Nej1

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atmire.migration.oldid1073
dc.contributor.advisorLees-Miller, Susan P.
dc.contributor.advisorCobb, Jennifer A.
dc.contributor.authorMahaney, Brandi Lynn
dc.date.accessioned2013-07-09T18:35:47Z
dc.date.available2015-07-10T07:00:28Z
dc.date.issued2013-07-09
dc.date.submitted2013en
dc.description.abstractDNA double-strand breaks (DSBs) are potentially toxic lesions. Maintenance of genomic stability requires the proper repair of DSBs by either the nonhomologous end-joining (NHEJ) pathway or the homologous recombination (HR) pathway. The XRCC4-like factor (XLF) and the NHEJ defective protein 1 (Nej1), stimulate the repair of DSBs by NHEJ in humans and Saccharomyces cerevisiae, respectively. The precise mechanism(s) through which XLF/Nej1 functions remain poorly understood. I show that the C-terminal region (CTR) of XLF is required for DNA binding and contains a conserved phenylalanine that is important for its interaction with Ku70/80 and for recruitment of XLF to DSBs in vivo. Surprisingly, I find that loss of the XLF-Ku interaction does not affect repair as measured by γ-H2AX foci resolution in vivo. In contrast, I show that while the CTR of the XLF homolog in S. cerevisiae, Nej1, is not required for recruitment to DSBs in vivo, it is critical for NHEJ in vivo. Previous studies by other groups have demonstrated a crucial role for the interaction between XLF and X-ray repair cross-complementing protein 4 (XRCC4) for NHEJ in vitro and in vivo. Similarly, I find that the interaction between Nej1 and the S. cerevisiae homolog of XRCC4, ligase interacting factor 1 (Lif1), correlates with NHEJ activity in vivo. These results bolster the current model of XLF function in which XLF/Nej1 stimulates NHEJ through its interaction with XRCC4/Lif1. In addition, my results uncover important differences in how XLF and Nej1 provide this function and provide useful tools for future investigation of the precise structural mechanism underlying their stimulation of NHEJ. I have also uncovered novel genetic interactions between an uncharacterized yeast gene, maintenance of telomere capping 5 (MTC5), the HR factor RAD52, and NHEJ factors including NEJ1. I show that MTC5 does not regulate NHEJ, but does regulate telomere length and dNTP levels. Based on a recent report and previously reported genetic interactions involving MTC5, I propose that the telomere length defects observed in mtc5Δ cells and the genetic interactions between MTC5, RAD52, and NHEJ factors may result from decreased dNTP levels and defects in amino acid uptake.en_US
dc.description.embargoterms2 yearsen_US
dc.identifier.citationMahaney, B. L. (2013). Physical and genetic interactions involving the nonhomologous end-joining factor XLF/Nej1 (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/27347en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/27347
dc.identifier.urihttp://hdl.handle.net/11023/776
dc.language.isoeng
dc.publisher.facultyGraduate Studies
dc.publisher.institutionUniversity of Calgaryen
dc.publisher.placeCalgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subjectChemistry--Cell
dc.subjectBiology--Molecular
dc.subjectBiochemistry
dc.subjectBiology--Molecular
dc.subject.classificationDNA repairen_US
dc.subject.classificationXLFen_US
dc.subject.classificationNej1en_US
dc.subject.classificationNHEJen_US
dc.titlePhysical and genetic interactions involving the nonhomologous end-joining factor XLF/Nej1
dc.typedoctoral thesis
thesis.degree.disciplineBiochemistry and Molecular Biology
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
ucalgary.item.requestcopytrue
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