Role of PCNA-dependent stimulation of 3′-phosphodiesterase and 3′–5′ exonuclease activities of human Ape2 in repair of oxidative DNA damage

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis

A regenerative approach to canine osteoarthritis using allogeneic, adipose-derived mesenchymal stem cells. Safety results of a long-term follow-up

Mesenchymal stem cells (MSC) are emerging as an effective therapeutic tool in treating canine osteoarthritis (OA). In this report, we focused on the questions of whether MSC transplantation has long-term beneficial effects for the improvement in motion and also evaluated the safety of MSC injection. Visceral adipose tissue, a surgical waste obtained during routine ovariectomy served as a source of allogeneic MSCs and used to treat OA. Altogether, fifty-eight dogs were transplanted in the study suffering from OA in the elbow (42 animals), hip (5), knee (8), ankle (2), and hock (1). The effect of MSC transplantation was evaluated by the degree of lameness at a 4-5-years follow-up period based on the owners' subjective observations. The results showed that 83% of the OA patients improved or retained improvement in lameness. Clinical safety of the treatment was assessed by evaluating the coincidence of tumors or other diseases and other adverse reactions (such as local inflammation) after MSC cell therapy. Two incidences of local inflammation for <1 week at the site of injection were reported. No other adverse reactions were detected post-treatment. Sixteen dogs died during the study, 4 due to cancer and 12 due to other diseases, diagnosed by veterinarians. Overall, our survey suggests that MSC transplantation has long-term beneficial effects in reducing lameness. Moreover, no enrichment in a specific cause of death was observed in the transplanted animals, compared to reported literature. Our data suggest that MSC treatment could be an effective and safe long-term therapy for canine OA

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. © 2013 European Molecular Biology Organization

Estimating the Rotation Rate in the Vacuolar Proton-ATPase in Native Yeast Vacuolar Membranes

The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibi-tor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low limit estimates. To our knowledge this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and it is not fixed on a solid support, instead it is functioning in its native membrane environment

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

Acknowledgments We thank Szilvia Minorits for technical assistance. I.U. conceived and designed the project and wrote the manuscript. All authors participated in designing and performing the experiments, and analyzing the results. The authors declare no competing financial interests. This work was also supported by a grant from the National Research, Development and Innovation Office GINOP-2.3.2-15-2016-00001. Funding: This work was supported by Hungarian Science Foundation Grant OTKA 109521 and National Research Development and Innovation Office GINOP-2.3.2-15-2016-00001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Human Ape2 protein has a 3′–5′ exonuclease activity that acts preferentially on mismatched base pairs

DNA damage, such as abasic sites and DNA strand breaks with 3′-phosphate and 3′-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3′-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3′-phosphodiesterase and 3′–5′ exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3′–5′ exonuclease and 3′-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3′-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3′–5′ exonuclease

The effect of sequence context on spontaneous Polζ-dependent mutagenesis in Saccharomyces cerevisiae

The Polζ translesion synthesis (TLS) DNA polymerase is responsible for over 50% of spontaneous mutagenesis and virtually all damage-induced mutagenesis in yeast. We previously demonstrated that reversion of the lys2ΔA746 −1 frameshift allele detects a novel type of +1 frameshift that is accompanied by one or more base substitutions and depends completely on the activity of Polζ. These ‘complex’ frameshifts accumulate at two discrete hotspots (HS1 and HS2) in the absence of nucleotide excision repair, and accumulate at a third location (HS3) in the additional absence of the translesion polymerase Polη. The current study investigates the sequence requirements for accumulation of Polζ-dependent complex frameshifts at these hotspots. We observed that transposing 13 bp of identity from HS1 or HS3 to a new location within LYS2 was sufficient to recapitulate these hotspots. In addition, altering the sequence immediately upstream of HS2 had no effect on the activity of the hotspot. These data support a model in which misincorporation opposite a lesion precedes and facilitates the selected slippage event. Finally, analysis of nonsense mutation revertants indicates that Polζ can simultaneously introduce multiple base substitutions in the absence of an accompanying frameshift event

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ\u27s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases

Development of a large-scale pathogen screening test for the biosafety evaluation of canine mesenchymal stem cells

Background The action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches. Results The established nucleic acid-based assays displayed remarkable sensitivity, detecting 100–1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus. Conclusion In this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens