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Mechanism of DNA Homologous Recombination through Studies of DNA Sliding Clamps, Clamp Loaders, and DNA Polymerases
Li, Jian

2013, Doctor of Philosophy (PhD), Ohio University, Molecular and Cellular Biology (Arts and Sciences).
DNA double-strand breaks (DSBs) are introduced into the genome by several factors including ionizing radiation, mutagenic chemicals, reactive oxygen species, and stalled DNA replication. Without appropriate repair, DSBs may lead to cell lethality or cancer. Homologous recombination (HR) is a widely conserved essential mechanism for high-fidelity repair of DSBs. Available evidence suggests that DNA sliding clamps and DNA polymerases are playing important roles in HR, but the biochemical details of their roles are unclear. This study focused on the functions of two DNA sliding clamps, proliferating cell nuclear antigen (PCNA) and the DNA damage checkpoint clamp (9-1-1 complex), and three DNA polymerases (Pol), Pol delta, Pol eta, and Pol zeta in HR processes. PCNA is required for DNA homologous recombination, but its exact role is unclear. The loading of PCNA onto a synthetic D-loop intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and Pol delta, eta, and zeta were investigated. PCNA was loaded onto the synthetic D-loop as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A (RPA), which is associated with the displaced single-stranded DNA loop and provides a binding site for the clamp-loader Replication Factor C (RFC). Loaded PCNA greatly stimulates DNA synthesis by Pol delta within the D-loop, but does not affect primer recognition by Pol delta. This suggests that the essential role of PCNA in HR is not recruitment of Pol delta to the D-loop, but is stimulation of Pol delta to displace a DNA strand during D-loop extension. Both Pol eta and Pol zeta extended the D-loop more efficiently than Pol delta in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Rad51 strongly inhibited both the loading of PCNA onto the D-loop and the extension of the D-loop by Pol delta and eta. However, pre-loaded PCNA on the D-loop counteracts the Rad51-mediated inhibition of the D-loop extension. This suggests that the inhibition of post-invasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading. Even though the 9-1-1 complex has been suggested to be potentially involved the post invasion synthesis of HR, neither its loading on the DNA substrate nor the stimulation of polymerase activity were detected in this study. This study contributes to our understanding of the mechanisms of HR-mediated DNA repair and maintenance of genome integrity.
Tomohiko Sugiyama (Advisor)
208 p.

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Li, J. (2013). Mechanism of DNA Homologous Recombination through Studies of DNA Sliding Clamps, Clamp Loaders, and DNA Polymerases. (Electronic Thesis or Dissertation). Retrieved from https://etd.ohiolink.edu/

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Li, Jian. "Mechanism of DNA Homologous Recombination through Studies of DNA Sliding Clamps, Clamp Loaders, and DNA Polymerases." Electronic Thesis or Dissertation. Ohio University, 2013. OhioLINK Electronic Theses and Dissertations Center. 04 Sep 2015.

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Li, Jian "Mechanism of DNA Homologous Recombination through Studies of DNA Sliding Clamps, Clamp Loaders, and DNA Polymerases." Electronic Thesis or Dissertation. Ohio University, 2013. https://etd.ohiolink.edu/

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Li, Jian accepted Dissertation 07-23-13 Su 13.pdf (13.99 MB) View|Download