Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair

Veteran Perspectives on Phantom Limb Pain and Non-Drug Interventions

Tonya L Rich,1,2 Hannah L Phelan,1,3 Amy A Gravely,1 Kierra J Falbo,1,2 Jacob A Finn,1,4 Mary E Matsumoto,1,5 Katherine J Muschler,1 Christine M Olney,1,2,6 Jessica E Kiecker,1 Andrew H Hansen1,2,7 1Minneapolis Veteran’s Affairs Health Care System, Minneapolis, MN, USA; 2Department of Rehabilitation Medicine, Division of Rehabilitation Science, University of Minnesota, Minneapolis, MN, USA; 3Medical College of Wisconsin, Milwaukee, WI, USA; 4Department of Psychiatry, University of Minnesota, Minneapolis, MN, USA; 5Department of Rehabilitation Medicine, University of Minnesota, Minneapolis, MN, USA; 6Department of Nursing, University of Minnesota, Minneapolis, MN, USA; 7Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USACorrespondence: Tonya L Rich, Tel +1 612-467-5463, Fax +1 612-725-2093, Email [email protected]: Phantom limb pain (PLP) commonly occurs post-amputation and can negatively affect the daily functioning of persons with amputation. Best practices for medication and non-drug management remain unclear.Objective: To better understand the PLP experience and patients’ familiarity with treatments, phone interviews were conducted at the Minneapolis Veterans Affairs Regional Amputation Center in Veterans with amputations.Methods: Fifty Veteran participants (average age 66, 96% male) with lower limb amputation were recruited for phone-based data collection of patient-reported outcomes (ie, demographics using the Trinity Amputation and Prosthesis Experience Scales-Revised (TAPES-R) and pain experience using the Phantom Phenomena Questionnaire) to characterize the population and a semi-structured interview. Notes taken during interviews were analyzed using the Krueger and Casey constant comparison analysis method.Results: Participants had an average of 15 years since amputation, and 80% reported PLP as identified with the Phantom Phenomena Questionnaire. Investigators identified several core themes from the qualitative interviews including 1) high variability in the experience of PLP, 2) acceptance and resilience, and 3) PLP treatment perceptions. The majority of participants reported trying common non-drug treatments with none endorsed consistently as highly effective.Conclusion: More research is needed to inform identification and implementation of clinical best practices for non-drug interventions for PLP and understand the factors that influence engagement in non-drug interventions. The participants in this study were largely male, so these results may not be generalizable to females.Keywords: amputation, phantom limb pain, rehabilitation, non-drug interventions, pai