Deletion of ULS1 confers damage tolerance in sgs1 mutants through a Top3-dependent D-loop mediated fork restart pathway |
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| Affiliation: | 1. Department of Pathology and Laboratory Medicine, Philadelphia, Pennsylvania, 19104, United States;2. Cell and Molecular Biology Group, Biomedical Graduate Studies, Philadelphia, Pennsylvania, 19104, United States;3. The Institute of Aging, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104, United States;1. Virgina Tech Carilion Research Institute, Virginia Tech, Roanoke, Virginia, 24016, USA;2. Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, VA, 24061, USA;3. Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, 16802, USA;4. Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA;5. Center for Structural Oncology, Pennsylvania State University, University Park, PA, 16802, USA;6. Comprehensive Cancer Center, Wake Forest School of Medicine, Wake Forest, NC, 27157, USA;1. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada;2. Department of Biochemistry, McGill University, Montreal, QC, Canada;1. Apartado Postal 218, Masaya, Nicaragua;1. Schmid College of Science, Chapman University, Orange, CA 92866, USA;2. Isaac Newton Institute, University of Cambridge, Cambridge, CB3 0EH, United Kingdom;3. Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA |
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| Abstract: | Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops. |
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| Keywords: | Top3 Uls1 Homologous recombination D-loop Hydroxyurea rDNA |
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