Limei Ju

PARP-3 and APLF Function Together to Accelerate Nonhomologous End-Joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.

TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo.

Defective Tyrosyl‐DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single‐strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1−/− mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1−/− mice display an inability to rapidly repair DNA SSBs associated with Top1–DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age‐dependent and progressive cerebellar atrophy. Tdp1−/− mice treated with topotecan, a drug that increases levels of Top1–DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1‐associated DNA strand breaks.

Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.

TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo.

Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3′- or 5′-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase ‘poisons’. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3′-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3′-tyrosyl DNA phosphodiesterase (3′-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1−/− cells, suggesting that Tdp2 contributes to cellular 3′-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1−/−/Tdp2−/−/− DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1−/− DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.

Requirement for DNA Ligase IV during Embryonic Neuronal Development

The embryonic ventricular and subventricular zones (VZ/SVZ) contain the neuronal stem and progenitor cells and undergo rapid proliferation. The intermediate zone (IZ) contains nonreplicating, differentiated cells. The VZ/SVZ is hypersensitive to radiation-induced apoptosis. Ablation of DNA non-homologous end-joining (NHEJ) proteins, XRCC4 or DNA ligase IV (LigIV), confers ataxia telangiectasia mutated (ATM)-dependent apoptosis predominantly in the IZ. We examine the mechanistic basis underlying these distinct sensitivities using a viable LigIV (Lig4Y288C) mouse, which permits an examination of the DNA damage responses in the embryonic and adult brain. Via combined analysis of DNA breakage, apoptosis, and cell-cycle checkpoint control in tissues, we show that apoptosis in the VZ/SVZ and IZ is activated by low numbers of DNA double-strand breaks (DSBs). Unexpectedly, high sensitivity in the VZ/SVZ arises from sensitive activation of ATM-dependent apoptosis plus an ATM-independent process. In contrast, the IZ appears to be hypersensitive to persistent DSBs. NHEJ functions efficiently in both compartments. The VZ/SVZ and IZ regions incur high endogenous DNA breakage, which correlates with VZ proliferation. We demonstrate a functional G2/M checkpoint in VZ/SVZ cells and show that it is not activated by low numbers of DSBs, allowing damaged VZ/SVZ cells to transit into the IZ. We propose a novel model in which microcephaly in LIG4 syndrome arises from sensitive apoptotic induction from persisting DSBs in the IZ, which arise from high endogenous breakage in the VZ/SVZ and transit of damaged cells to the IZ. The VZ/SVZ, in contrast, is highly sensitive to acute radiation-induced DSB formation.

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.

ABSTRACT The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), ataxia telangiectasia mutated (Atm−/−) and double mutant Atm−/−/Lig4Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice. Summary: This study shows that endogenous and radiation-induced DNA damage sensitively activates apoptosis in the adult sub-ventricular zone (SVZ). Developmentally regulated apoptosis is shown in the neonatal SVZ.

A coordinated DNA damage response promotes adult quiescent neural stem cell activation

Stem and differentiated cells frequently differ in their response to DNA damage, which can determine tissue sensitivity. By exploiting insight into the spatial arrangement of subdomains within the adult neural subventricular zone (SVZ) in vivo, we show distinct responses to ionising radiation (IR) between neural stem and progenitor cells. Further, we reveal different DNA damage responses between neonatal and adult neural stem cells (NSCs). Neural progenitors (transit amplifying cells and neuroblasts) but not NSCs (quiescent and activated) undergo apoptosis after 2 Gy IR. This response is cell type- rather than proliferation-dependent and does not appear to be driven by distinctions in DNA damage induction or repair capacity. Moreover, exposure to 2 Gy IR promotes proliferation arrest and differentiation in the adult SVZ. These 3 responses are ataxia telangiectasia mutated (ATM)-dependent and promote quiescent NSC (qNSC) activation, which does not occur in the subdomains that lack progenitors. Neuroblasts arising post-IR derive from activated qNSCs rather than irradiated progenitors, minimising damage compounded by replication or mitosis. We propose that rather than conferring sensitive cell death, apoptosis is a form of rapid cell death that serves to remove damaged progenitors and promote qNSC activation. Significantly, analysis of the neonatal (P5) SVZ reveals that although progenitors remain sensitive to apoptosis, they fail to efficiently arrest proliferation. Consequently, their repopulation occurs rapidly from irradiated progenitors rather than via qNSC activation.