Aman Seth

End-processing during non-homologous end-joining: a role for exonuclease 1

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5′-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.

Recipient Myeloid-Derived Immunomodulatory Cells Induce PD-1 Ligand–Dependent Donor CD4+Foxp3+ Regulatory T Cell Proliferation and Donor–Recipient Immune Tolerance after Murine Nonmyeloablative Bone Marrow Transplantation

We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor–recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4–dependent expansion of donor Foxp3+ naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2d) hosts were administered TLI/ATS and BMT from WT or STAT6−/− C57BL/6 (H-2b) donors. Following STAT6−/− BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4–responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b+ cell subsets (Gr-1highCD11c−, Gr-1intCD11c−, and Gr-1lowCD11c+) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1lowCD11c+ cells induced potent H-2Kb+CD4+Foxp3+ nTreg proliferation in vitro in 72-h MLRs. Gr-1lowCD11c+ cells were reduced significantly in STAT6−/− and iNKT cell–deficient Jα18−/− BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to Jα18−/− BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3+ nTreg proliferation and protection from CD8 effector T cell–mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-β signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1lowCD11c+ cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand–dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.

Repair of Topoisomerase II-Mediated DNA Damage: Fixing DNA Damage Arising from a Protein Covalently Trapped on DNA

The generation of elevated levels of enzyme-DNA covalent complexes is the key event in cell killing by many drugs targeting DNA topoisomerases. These agents, termed topoisomerase poisons generate protein-linked DNA strand that block transcription and replication, leading to cell death. Cells treated with topoisomerase targeting drugs rely on diverse repair pathways to maintain genome stability and cell survival. This chapter summarizes current information on how DNA damage arising from Top2 poisons is repaired. A critical and unique area of repair of Top2 damage is the removal of protein that is covalently attached to DNA. Several specialized repair enzymes have recently been shown to remove Top2 covalently bound to DNA including TTRAP/Tdp2, CtIP, and the MRN (Mre11/Rad50/Nbs1) complex. New genetic approaches have led to the identification of many other genes important for repairing Top2 generated DNA damage. These newly identified genes will be important in understanding the repair processes that occur with this unique type of DNA damage, and promise to provide new markers for predicting the therapeutic efficacy of this important class of anticancer drugs.

Bidirectional immune tolerance in non-myeloablative MHC-mismatched BMT for murine β-thalassemia

Nonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4-driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand-expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and β-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

UNIT 3.3 Topoisomerase Assays

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro. Curr. Protoc. Pharmacol. 57:3.3.1-3.3.27.