The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.Oxidative DNA damage is generated at high levels in mammalian cells, even in cells not exposed to exogenous sources of reactive oxygen species. Several kinds of DNA modifications are formed upon oxidative stress (8). The most prevalent modifications, quantitatively, are single-strand breaks and oxidized bases. Clustered DNA damage, when two or more modifications are closely positioned in opposite strands, is detectable after gamma irradiation and has recently been shown to be generated by normal oxidative metabolism (3, 35). One unique aspect of such clustered lesions is that they can be converted into double-strand breaks (DSB) if a DNA glycosylase removes the two opposite bases and an apurinic/apyrimidinic (AP)-endonuclease cleaves the resulting abasic sites. Thus, although quantitatively minor, DSB are possible outcomes of oxidative DNA damage.Oxidized DNA bases are repaired primarily by the base excision repair pathway (BER) (22, 39). BER is initiated by a lesion-specific DNA N-glycosylase that recognizes and excises the damaged base. Eight-hydroxyguanine (8-oxoG) is one of the most abundant oxidized bases detected in cellular DNA. This adduct is easily bypassed by replicative polymerases; however, it can direct the misincorporation of adenine opposite 8-oxoG, thus leading to G·C-to-T·A transversion mutations (31). 8-oxoG accumulation has been causally associated with carcinogenesis and aging in several experimental models (1, 12). In eukaryotes, oxoguanine DNA glycosylase (OGG1) is the major 8-oxoG DNA glycosylase. OGG1 possesses an associated AP-lyase activity, such that it removes 8-oxoG and cleaves the DNA backbone. Human cells express two distinct OGG1 isoforms, α and β, which share the first 316 amino acids but differ significantly in their C termini (25). While OGG1-α is a bone fide DNA glycosylase (5) and localizes both to nuclei and mitochondria, OGG1-β localizes exclusively to mitochondria. We recently showed that the recombinant OGG1-β protein has no DNA glycosylase activity (13). The high degree of conservation of repair pathways for 8-oxoG, from bacteria to humans, along with epidemiological data correlating OGG1 polymorphisms and activity with predisposition to some cancers (11, 27, 33) attest to the biological importance of the repair of 8-oxoGs and other oxidative DNA lesions.Until recently, distinct classes of DNA lesions were believed to be metabolized by different and independent repair pathways. However, experimental evidence indicates that these pathways can interact and that there is a considerable degree of overlap in their substrate specificity and in the proteins that participate in each pathway. Experiments using yeast strains lacking one or more distinct DNA repair genes suggest that DSB repair pathways may play a role in repair of oxidative DNA damage. Swanson et al. showed that while yeast cells lacking ntg1 and ntg2 (homologues of Escherichia coli endonuclease III, a DNA glycosylase specific for pyrimidine lesions formed by oxidation) and apn1 (the major yeast abasic site endonuclease) are not overtly sensitive to oxidative stress, the additional disruption of the rad52 gene significantly increases sensitivity to H₂O₂ and menadione (36). Similarly, yeast cells expressing decreased levels of frataxin, which leads to elevated oxidative stress, show accumulation of oxidative damage in nuclear DNA only in a rad52 mutant background (18). RAD52 is a member of the RAD51 epistatic group. These proteins are believed to be involved in the early steps of homologous recombination, contributing to homology search and strand invasion; disruption of the corresponding genes renders cells deficient in DSB repair and hyper-recombinogenic (19).These results suggested a possible role for RAD52 in the repair of oxidative DNA damage. Moreover, an in vitro screening of protein partners that interact physically with OGG1-β performed in our lab (unpublished data) showed that human RAD52 strongly interacted with this glycosylase, again suggesting a possible function for RAD52 in the oxidative DNA damage response. Thus, we investigated whether RAD52 plays a role in the repair of oxidative DNA damage in human cells. We show here that human RAD52 physically interacts with both OGG1-α and -β, in vitro and in cell extracts. We also show that OGG1-α and -β inhibit RAD52 enzymatic activities. Conversely, RAD52 stimulates OGG1-α 8-oxoG incision activity. RAD52 colocalizes with OGG1-α in cells, and this colocalization increases after oxidative stress. Moreover, lower RAD52 expression, via gene knockdown (KD) or disruption of the RAD52 gene, render cells sensitive to oxidative stress. Based on our results, we discuss a model in which OGG1 and RAD52 cooperate to repair 8-oxoG lesions.     

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.Subject terms: CNS cancer, DNA damage and repair, Cell death

The complex effects of combined CDki/SPyADI application on arginine-auxotrophic glioblastoma multiforme cells. CDki/SPyADI combination therapy impairs cell proliferation, invasiveness, gene expression, induces mitochondrial impairment, and vacuole formation. Abemaciclib-SPyADI-treatment suppresses DNA repair, dinaciclib-SpyADI-treatment enhances γ-H2AX accumulation and activates the stress sensor GADD45 and β-catenin antagonist AXIN2. Both CDKi/SpyADI combinations significantly boost cell death.     

Incorporation of calcium into the soft tissues and calcareous corpuscles of larval Taenia taeniaeformis.

Larval Taenia taeniaeformis in vivo accumulates 45Ca2+ in soft tissues and calcareous corpuscles. Radioactivity was demonstrable in the corpuscles six months after a single dose of 45Ca2+ was administered to the host by means of a stomach tube. Ca2+ also was taken up by isolated larvae. Accumulation in vitro was more rapid then in vivo and was correlated with the external Ca2+ concentration. Temperature variation, oxygen availability, and metabolic inhibitors had little effect on the Ca2+ uptake, indicating that active transport of Ca2+ is unlikely in this parasite. Variations in the external Pi concentrations had no effect on Ca2+ accumulation or on its distribution. Addition of 5% CO2 increased the uptake of Ca2+ by the calcareous corpuscles under anaerobic conditions. Radioactivity from NaH14CO3 also was accumulated in soft tissues and corpuscles of T. taeniaeformis. Assuming that the 14C taken up by the corpuscles was in the form of 14CO3(2-), the ratio of Ca2+ to CO3(2-) accumulation in the corpuscles approximates the ratio of these constituents in dolomite: CaMg(CO3)2.     

The activity of tricyclic antidepressant drugs against Trypanosoma cruzi

Imipramine and related derivatives were tested as possible chemotherapeutic agents against Trypanosoma cruzi parasites in vitro. The IC50 values and the lethal concentrations for two cloned stocks of the parasite were determined. 2-Nitrodesmethylimipramine was the most effective compound tested (IC50 = 4-7 microM). Parasites that were able to grow and to complete the intracellular cycle in mammalian cells in the presence of the drug could be selected. Differences in susceptibility to some imipramine analogs between T. cruzi-cloned stocks were found. The study also shows that modification of the imipramine molecule by electron-withdrawing groups greatly enhances its biological activity.     

SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.     

The acoustic effect of vocal tract adjustments in zebra finches

Vocal production in songbirds requires the control of the respiratory system, the syrinx as sound source and the vocal tract as acoustic filter. Vocal tract movements consist of beak, tongue and hyoid movements, which change the volume of the oropharyngeal–esophageal cavity (OEC), glottal movements and tracheal length changes. The respective contributions of each movement to filter properties are not completely understood, but the effects of this filtering are thought to be very important for acoustic communication in birds. One of the most striking movements of the upper vocal tract during vocal behavior in songbirds involves the OEC. This study measured the acoustic effect of OEC adjustments in zebra finches by comparing resonance acoustics between an utterance with OEC expansion (calls) and a similar utterance without OEC expansion (respiratory sounds induced by a bilateral syringeal denervation). X-ray cineradiography confirmed the presence of an OEC motor pattern during song and call production, and a custom-built Hall-effect collar system confirmed that OEC expansion movements were not present during respiratory sounds. The spectral emphasis during zebra finch call production ranging between 2.5 and 5 kHz was not present during respiratory sounds, indicating strongly that it can be attributed to the OEC expansion.     

HIPK2: A tumour suppressor that controls DNA damage‐induced cell fate and cytokinesis

In response to DNA‐damage, cells have to decide between different cell fate programmes. Activation of the tumour suppressor HIPK2 specifies the DNA damage response (DDR) and tips the cell fate balance towards an apoptotic response. HIPK2 is activated by the checkpoint kinase ATM, and triggers apoptosis through regulatory phosphorylation of a set of cellular key molecules including the tumour suppressor p53 and the anti‐apoptotic corepressor CtBP. Recent work has identified HIPK2 as a regulator of the ultimate step in cytokinesis: the abscission of the mother and daughter cells. Since proper cytokinesis is essential for genome stability and maintenance of correct ploidy, this finding sheds new light on the tumour suppressor function of HIPK2. Here we highlight the molecular mechanisms coordinating HIPK2 function and discuss its emerging role as a tumour suppressor.