Single-Molecule Imaging of DNA Organization and Repair
Graham, Thomas G.W.
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AbstractIn this dissertation, I describe two applications of single-molecule fluorescence imaging for the study of protein-DNA interactions involved in chromosome segregation and DNA double- strand break repair.
Chromosome segregation in some bacteria requires the formation of centromere-like complexes by the ParB protein, which binds specifically to parS sites near the origin of replication and “spreads” over multiple kilobases of adjacent nonspecific DNA. The mechanism of this spreading is poorly understood. Using single-molecule fluorescence imaging of flow-stretched DNA molecules, we demonstrated that the bacterial centromere-associated protein Spo0J (ParB) is able to bridge different segments of DNA. We identified bridging-deficient mutants of Spo0J and showed that they were impaired for spreading in vivo. These results argue that DNA bridging is required for ParB spreading.
Non-homologous end joining (NHEJ) is the major mechanism of DNA double-strand break repair in vertebrate cells. Though the components of the NHEJ pathway have been characterized genetically and biochemically, it has been unclear how these components cooperate to form a complex that holds together DNA ends for processing and ligation. We showed that an extract from the eggs of the frog Xenopus laevis joins DNA ends in a manner dependent on the major components of the NHEJ pathway. We developed single molecule fluorescence colocalization and Förster resonance energy transfer (FRET) assays for monitoring bridging of DNA ends during NHEJ in egg extract. Our results reveal two stages of end bridging: Ends are initially tethered, but not closely aligned, in a “long-range” complex requiring the Ku70/80 and DNA-PKcs proteins. Transition to a “short-range” complex, in which the ends are held closely together, requires DNA- PK catalytic activity as well as XLF and the LIG4:XRCC4 complex. Possible implications are discussed for the regulation of DNA end processing.
As an extension of this work, we have used our bulk and single-molecule NHEJ assays to investigate the role of the XLF protein in end joining. In contrast to a popular model of XLF-XRCC4 filament formation, preliminary evidence suggests that only a single XLF dimer binds to the NHEJ synaptic complex shortly prior to its transition to the short-range complex. Furthermore, analysis of synthetic XLF heterodimers indicates that interaction with XRCC4 on only one face of the XLF dimer is sufficient for end joining. Our results suggest that rather than forming filaments with XRCC4 to tether DNA ends, XLF instead plays a structural or allosteric role in the transition of the synaptic complex to a short-range state.
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