Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Frederick B. Johnson
Homologous recombination (HR), becomes important for repair during replication where completion of DNA synthesis relies on recombination intermediate-mediated lesion bypass. For decades, Holliday Junctions (HJs) were considered the primary recombination intermediate utilized during this repair process, but increasing evidence points out two strong discrepancies: 1) X-structures, when present, are often biochemically inconsistent with being HJs, and 2) despite HR mutants being sensitive to numerous DNA damaging agents, most insults don’t result in X-structure accumulation, suggesting alternative HR pathways are at play. The Sgs1-Top3-Rmi1 (STR) complex, in Saccharomyces cerevisiae, is vital for maintaining genome integrity, and is known to resolve recombination intermediates. We took advantage of this function to identify the recombination intermediates employed, and consequently, the distinct HR pathways at play in response to damage induced by methyl methanesulfonate (MMS), and the ribonucleotide reductase inhibitor, hydroxyurea.
Using genetic manipulation, 2D-gel electrophoresis and in vitro biochemical and enzymatic assays, we find that Top3, unassisted by Sgs1 and Rmi1, is able to function through two distinct HR-dependent mechanisms. In the presence of MMS, Top3 is able to provide rescue and reduce recombination-dependent X-structures in sgs1Δ mutants. We find that these X-structures are biochemically consistent with being hemicatenane-related template switch recombination intermediates (Rec-Xs) and not HJs. Furthermore, Top3 and the entire STR complex are capable of resolving Rec-Xs but not equivalent dHJs in vitro. We also find that in the absence of the sumo-targeted ubiquitin ligase, Uls1, Top3 provides rescue to sgs1Δ mutants on HU through a mechanism entirely distinct from Rec-X resolution. We show that in the absence of Sgs1 and Uls1, Top3 is required to promote site specific breaks, and subsequent Rad51-dependent D-loop-mediated fork restart, distinct from a Rad51-independent mechanism in sgs1Δ mutants, and indicative of a Uls1-mediated repair pathway switch. These activities point to the use of template-switch recombination (via gap repair) as a primary mechanism for bypassing MMS-induced damage and coordinated break and D-loop-meditated fork reestablishment in response to HU-induced fork stalling. Importantly, this work highlights novel differences between primary HR repair mechanisms in response to different types of DNA damage, and provides evidence for context-dependent repair pathway choice.
Glineburg, Mary Rebecca, "Dna Disentangled: Roles For Sgs1 And Top3 In Rec-X Resolution And Replication Fork Restart" (2016). Publicly Accessible Penn Dissertations. 2311.