Proteomic analysis reveals mechanisms underlying increased efficacy of bleomycin by photochemical internalization in bladder cancer cells.

Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.

Long-term Effectiveness of Ethanol Ablation in Controlling Neck Nodal Metastases in Childhood Papillary Thyroid Cancer.

Context: Childhood papillary thyroid carcinoma (CPTC), despite bilateral thyroidectomy, nodal dissection and radioiodine remnant ablation (RRA), recurs within neck nodal metastases (NNM) in 33% within 20 postoperative years. These NNM are usually treated with reoperation or further radioiodine. Ethanol ablation (EA) may be considered when numbers of NNM are limited. Objective: We studied the long-term results of EA in 14 patients presenting with CPTC during 1978 to 2013 and having EA for NNM during 2000 to 2018. Methods: Cytologic diagnoses of 20 NNM (median diameter 9 mm; median volume 203 mm3) were biopsy proven. EA was performed during 2 outpatient sessions under local anesthesia; total volume injected ranged from 0.1 to 2.8 cc (median 0.7). All were followed regularly by sonography and underwent volume recalculation and intranodal Doppler flow measurements. Successful ablation required reduction both in NNM volume and vascularity. Results: Post EA, patients were followed for 5 to 20 years (median 16). There were no complications, including postprocedure hoarseness. All 20 NNM shrank (mean by 87%) and Doppler flow eliminated in 19 of 20. After EA, 11 NNM (55%) disappeared on sonography; 8 of 11 before 20 months. Nine ablated foci were still identifiable after a median of 147 months; only one identifiable 5-mm NNM retained flow. Median serum Tg post EA was 0.6 ng/mL. Only one patient had an increase in Tg attributed to lung metastases. Conclusion: EA of NNM in CPTC is effective and safe. Our results suggest that for CPTC patients who do not wish further surgery and are uncomfortable with active surveillance of NNM, EA represents a minimally invasive outpatient management option.

PCNA regulates primary metabolism by scaffolding metabolic enzymes.

The essential roles of proliferating cell nuclear antigen (PCNA) as a scaffold protein in DNA replication and repair are well established, while its cytosolic roles are less explored. Two metabolic enzymes, alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), both contain PCNA interacting motifs. Mutation of the PCNA interacting motif APIM in ENO1 (F423A) impaired its binding to PCNA and resulted in reduced cellular levels of ENO1 protein, reduced growth rate, reduced glucose consumption, and reduced activation of AKT. Metabolome and signalome analysis reveal large consequences of impairing the direct interaction between PCNA and ENO1. Metabolites above ENO1 in glycolysis accumulated while lower glycolytic and TCA cycle metabolite pools decreased in the APIM-mutated cells; however, their overall energetic status were similar to parental cells. Treating haematological cancer cells or activated primary monocytes with a PCNA targeting peptide drug containing APIM (ATX-101) also lead to a metabolic shift characterized by reduced glycolytic rate. In addition, we show that ATX-101 treatments reduced the ENO1 - PCNA interaction, the ENO1, GAPDH and 6PGD protein levels, as well as the 6PGD activity. Here we report for the first time that PCNA acts as a scaffold for metabolic enzymes, and thereby act as a direct regulator of primary metabolism.

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue.

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2 O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 µg/mm2 . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.

UDP-glucose dehydrogenase expression is upregulated following EMT and differentially affects intracellular glycerophosphocholine and acetylaspartate levels in breast mesenchymal cell lines.

Metabolic rewiring is one of the indispensable drivers of epithelial-mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knock-down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knock-down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA-mediated knock-down of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.

Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis.

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation.

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA.

BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells.

There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2. Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2.

Cancer-induced muscle atrophy is determined by intrinsic muscle oxidative capacity.

We tested the hypothesis that cancer cachexia progression would induce oxidative post-translational modifications (Ox-PTMs) associated with skeletal muscle wasting, with different responses in muscles with the prevalence of glycolytic and oxidative fibers. We used cysteine-specific isotopic coded affinity tags (OxICAT) and gel-free mass spectrometry analysis to investigate the cysteine Ox-PTMs profile in the proteome of both plantaris (glycolytic) and soleus (oxidative) muscles in tumor-bearing and control rats. Histological analysis revealed muscle atrophy in type II fibers in plantaris muscle, with no changes in plantaris type I fibers and no differences in both soleus type I and II fibers in tumor-bearing rats when compared to healthy controls. Tumor progression altered the Ox-PTMs profile in both plantaris and soleus. However, pathway analysis including the differentially oxidized proteins revealed tricarboxylic acid cycle and oxidative phosphorylation as main affected pathways in plantaris muscle from tumor-bearing rats, while the same analysis did not show main metabolic pathways affected in the soleus muscle. In addition, cancer progression affected several metabolic parameters such as ATP levels and markers of oxidative stress associated with muscle atrophy in plantaris muscle, but not in soleus. However, isolated soleus from tumor-bearing rats had a reduced force production capacity when compared to controls. These novel findings demonstrate that tumor-bearing rats have severe muscle atrophy exclusively in glycolytic fibers. Cancer progression is associated with cysteine Ox-PTMs in the skeletal muscle, but these modifications affect different pathways in a glycolytic muscle compared to an oxidative muscle, indicating that intrinsic muscle oxidative capacity determines the response to cancer cachectic effects.

Exercise training reverses cancer-induced oxidative stress and decrease in muscle COPS2/TRIP15/ALIEN.

OBJECTIVE: We tested the hypothesis that exercise training would attenuate metabolic impairment in a model of severe cancer cachexia. METHODS: We used multiple in vivo and in vitro methods to explore the mechanisms underlying the beneficial effects induced by exercise training in tumor-bearing rats. RESULTS: Exercise training improved running capacity, prolonged lifespan, reduced oxidative stress, and normalized muscle mass and contractile function in tumor-bearing rats. An unbiased proteomic screening revealed COP9 signalosome complex subunit 2 (COPS2) as one of the most downregulated proteins in skeletal muscle at the early stage of cancer cachexia. Exercise training normalized muscle COPS2 protein expression in tumor-bearing rats and mice. Lung cancer patients with low endurance capacity had low muscle COPS2 protein expression as compared to age-matched control subjects. To test whether decrease in COPS2 protein levels could aggravate or be an intrinsic compensatory mechanism to protect myotubes from cancer effects, we performed experiments in vitro using primary myotubes. COPS2 knockdown in human myotubes affected multiple cellular pathways, including regulation of actin cytoskeleton. Incubation of cancer-conditioned media in mouse myotubes decreased F-actin expression, which was partially restored by COPS2 knockdown. Direct repeat 4 (DR4) response elements have been shown to positively regulate gene expression. COPS2 overexpression decreased the DR4 activity in mouse myoblasts, and COPS2 knockdown inhibited the effects of cancer-conditioned media on DR4 activity. CONCLUSIONS: These studies demonstrated that exercise training may be an important adjuvant therapy to counteract cancer cachexia and uncovered novel mechanisms involving COPS2 to regulate myotube homeostasis in cancer cachexia.

HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell lines.

BACKGROUND: HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used in the treatment of lymphocyte-derived malignancies, but their mechanisms of action remain poorly understood. Here we aimed to identify novel protein targets of HDACi in B- and T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines. METHODS: Jurkat T- and SUDHL5 B-lymphocytes were treated with the HDACi SAHA (vorinostat) prior to SILAC-based quantitative proteome analysis. Selected differentially expressed proteins were verified by targeted mass spectrometry, RT-PCR and western analysis in multiple mammalian cell lines. Genomic uracil was quantified by LC-MS/MS, cell cycle distribution analyzed by flow cytometry and class switch recombination monitored by FACS in murine CH12F3 cells. RESULTS: SAHA treatment resulted in differential expression of 125 and 89 proteins in Jurkat and SUDHL5, respectively, of which 19 were commonly affected. Among these were several oncoproteins and tumor suppressors previously not reported to be affected by HDACi. Several key enzymes determining the cellular dUTP/dTTP ratio were downregulated and in both cell lines we found robust depletion of UNG2, the major glycosylase in genomic uracil sanitation. UNG2 depletion was accompanied by hyperacetylation and mediated by increased proteasomal degradation independent of cell cycle stage. UNG2 degradation appeared to be ubiquitous and was observed across several mammalian cell lines of different origin and with several HDACis. Loss of UNG2 was accompanied by 30-40% increase in genomic uracil in freely cycling HEK cells and reduced immunoglobulin class-switch recombination in murine CH12F3 cells. CONCLUSION: We describe several oncoproteins and tumor suppressors previously not reported to be affected by HDACi in previous transcriptome analyses, underscoring the importance of proteome analysis to identify cellular effectors of HDACi treatment. The apparently ubiquitous depletion of UNG2 and PCLAF establishes DNA base excision repair and translesion synthesis as novel pathways affected by HDACi treatment. Dysregulated genomic uracil homeostasis may aid interpretation of HDACi effects in cancer cells and further advance studies on this class of inhibitors in the treatment of APOBEC-expressing tumors, autoimmune disease and HIV-1.

Absence of NLRP3 Inflammasome in Hematopoietic Cells Reduces Adverse Remodeling After Experimental Myocardial Infarction.

An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3 -/- mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1ß and -18) in MI patients.

A Novel Truncated Form of Nephronectin Is Present in Small Extracellular Vesicles Isolated from 66cl4 Cells.

Extracellular vesicles are emerging as biomarkers in breast cancer. Our recent report suggested that an intracellular granular staining pattern of the extracellular matrix protein nephronectin (NPNT) in breast tumor sections correlated with a poor prognosis. Furthermore, the results showed that NPNT is localized in extracellular vesicles derived from mouse breast cancer cells. In this study, we performed proteomic analysis that revealed that several proteins, including tumor-promoting molecules, are differentially expressed in the cargo of small extracellular vesicles (sEVs) derived from NPNT-expressing mouse breast cancer cells. We also identified three different forms of NPNT at 80, 60, and 20 kDa. We report that the native form of NPNT at 60 kDa becomes further glycosylated and is detected as the 80 kDa NPNT, which may be processed by matrix metalloproteinases to a shorter form of around 20 kDa, which has not previously been described. Although both 80 and 20 kDa NPNT are detected in sEVs derived from breast cancer cells, the 20 kDa form of NPNT is concentrated in sEVs. In summary, we show that a novel truncated form of NPNT is found in sEVs derived from breast cancer cells.

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy.

Receptor tyrosine kinases (RTKs), such as HER2 and/or EGFR are important therapeutic targets in multiple cancer cells. Low and/or short response to targeted therapies are often due to activation of compensatory signaling pathways, and therefore a combination of kinase inhibitors with other anti-cancer therapies have been proposed as promising strategies. PCNA is recently shown to have non-canonical cytosolic roles, and targeting PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM is shown to mediate changes in central signaling pathways such as PI3K/Akt and MAPK, acting downstream of multiple RTKs. In this study, we show how targeting PCNA increased the anti-cancer activity of EGFR/HER2/VEGFR inhibition in vitro as well as in vivo. The combination treatment resulted in reduced tumor load and increased the survival compared to either single agent treatments. The combination treatment affected multiple cellular signaling responses not seen by EGFR/HER2/VEGFR inhibition alone, and changes were seen in pathways determining protein degradation, ER-stress, apoptosis and autophagy. Our results suggest that targeting the non-canonical roles of PCNA in cellular signaling have the potential to improve targeted therapies.

Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity.

Given the association between high aerobic capacity and the prevention of metabolic diseases, elucidating the mechanisms by which high aerobic capacity regulates whole-body metabolic homeostasis is a major research challenge. Oxidative post-translational modifications (Ox-PTMs) of proteins can regulate cellular homeostasis in skeletal and cardiac muscles, but the relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism is still unclear. Here, we evaluated the Ox-PTM profile in cardiac and skeletal muscles of rats bred for low (LCR) and high (HCR) intrinsic aerobic capacity. Redox proteomics screening revealed different cysteine (Cys) Ox-PTM profile between HCR and LCR rats. HCR showed a higher number of oxidized Cys residues in skeletal muscle compared to LCR, while the opposite was observed in the heart. Most proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of oxidative metabolism. The most oxidized protein in the skeletal muscle of HCR rats was malate dehydrogenase (MDH1). HCR showed higher MDH1 activity compared to LCR in skeletal, but not cardiac muscle. These novel findings indicate a clear association between Cys Ox-PTMs and aerobic capacity, leading to novel insights into the role of Ox-PTMs as an essential signal to maintain metabolic homeostasis.

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes.

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

Marine-Lenhart syndrome in two adolescents, including one with thyroid cancer: a case series and review of the literature.

BACKGROUND: The coexistence of functional thyroid nodules and Graves' disease (GD) is a rare condition known as Marine-Lenhart syndrome. Thyroid cancer has been described in several adults, but never in children, with Marine-Lenhart syndrome. This paper discusses the challenges in diagnosis and the unique management of this condition in children, in the context of extant literature. CONTENT: In this case report, two adolescent female patients with Marine-Lenhart syndrome, aged 15 and 16 years, exhibited biochemical evidence of hyperthyroidism, and were found to have unilateral hyperfunctioning thyroid nodules via thyroid scintigraphy. Additionally, both patients showed elevated thyroid-stimulating immunoglobulins (TSI) and increased glandular activity, confirming background GD. Notably, one patient was also diagnosed with intranodular thyroid cancer upon preoperative examination. Both patients were treated via surgical resection. Summary and outlook: Diagnosis of Marine-Lenhart syndrome can be made in patients with functional thyroid nodules and increased glandular activity on thyroid scintigraphy. Standard doses of radioiodine ablation are not effective in the majority of patients and should be avoided due to the increased risk for thyroid cancer, making thyroidectomy the preferred treatment.

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process.