The bacterial DNA sliding clamp, ß-clamp: structure, interactions, dynamics and drug discovery.

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.

Characterization of POLE c.1373A > T p.(Tyr458Phe), causing high cancer risk.

The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP.

Peptides containing the PCNA interacting motif APIM bind to the ß-clamp and inhibit bacterial growth and mutagenesis.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the ß-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the ß-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial ß-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

Differentially Expressed Extracellular Vesicle-Contained microRNAs before and after Transurethral Resection of Bladder Tumors.

Bladder cancer (BC) is currently diagnosed and monitored by cystoscopy, a costly and invasive procedure. Potential biomarkers in urine, blood, and, more recently, extracellular vesicles (EVs), have been explored as non-invasive alternatives for diagnosis and surveillance of BC. EVs are nanovesicles secreted by most cell types containing diverse molecular cargo, including different types of small RNAs, such as microRNA (miRNA). In this study, we performed next-generation sequencing of EV-contained miRNA isolated from urine and serum of 41 patients with non-muscle invasive BC (27 stage Ta, 14 stage T1) and 15 non-cancer patients (NCP) with benign cystoscopy findings. MiRNA sequencing was also performed on serum supernatant samples for T1 patients. To identify potential BC-specific biomarkers, expression levels of miRNA in presurgery samples were compared to those at postsurgery check-ups, and to NCPs. Results showed that two miRNAs, urinary EV-contained miR-451a and miR-486-5p, were significantly upregulated in presurgery samples from T1 patients compared to postsurgery check-up samples. This was confirmed in a replica EV/RNA isolation and sequencing run of 10 T1 patients from the primary run; however, analyses revealed no differential expression of miRNAs in serum EVs, serum supernatant, or when comparing BC patients to NCPs. This is the first study to investigate EV-containing miRNA sequencing in pre- and postsurgery BC patient samples and our findings suggest that urinary EV-contained miR-451a and miR-486-5p may be potential biomarkers for recurrence-free survival of BC patients with stage T1 disease.

The PCNA interaction motifs revisited: thinking outside the PIP-box.

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.

Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA).

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT.

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.

APIM-Mediated REV3L⁻PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells.

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling.

Targeting proliferating cell nuclear antigen (PCNA) for cancer therapy.

Proliferating cell nuclear antigen (PCNA) is an essential scaffold protein in many cellular processes. It is best known for its role as a DNA sliding clamp and processivity factor during DNA replication, which has been extensively reviewed by others. However, the importance of PCNA extends beyond its DNA-associated functions in DNA replication, chromatin remodelling, DNA repair and DNA damage tolerance (DDT), as new non-canonical roles of PCNA in the cytosol have recently been identified. These include roles in the regulation of immune evasion, apoptosis, metabolism, and cellular signalling. The diverse roles of PCNA are largely mediated by its myriad protein interactions, and its centrality to cellular processes makes PCNA a valid therapeutic anticancer target. PCNA is expressed in all cells and plays an essential role in normal cellular homeostasis; therefore, the main challenge in targeting PCNA is to selectively kill cancer cells while avoiding unacceptable toxicity to healthy cells. This chapter focuses on the stress-related roles of PCNA, and how targeting these PCNA roles can be exploited in cancer therapy.

tRNA-Derived Fragments as Biomarkers in Bladder Cancer.

Bladder cancer (BC) diagnosis is reliant on cystoscopy, an invasive procedure associated with urinary tract infections. This has sparked interest in identifying noninvasive biomarkers in body fluids such as blood and urine. A source of biomarkers in these biofluids are extracellular vesicles (EVs), nanosized vesicles that contain a wide array of molecular cargo, including small noncoding RNA such as transfer RNA-derived fragments (tRF) and microRNA. Here, we performed small-RNA next-generation sequencing from EVs from urine and serum, as well as from serum supernatant. RNA was extracted from 15 non-cancer patients (NCPs) with benign findings in cystoscopy and 41 patients with non-muscle invasive BC. Urine and serum were collected before transurethral resection of bladder tumors (TUR-b) and at routine post-surgery check-ups. We compared levels of tRFs in pre-surgery samples to samples from NCPs and post-surgery check-ups. To further verify our findings, samples from 10 patients with stage T1 disease were resequenced. When comparing tRF expression in urine EVs between T1 stage BC patients and NCPs, 14 differentially expressed tRFs (DEtRFs) were identified. In serum supernatant, six DEtRFs were identified among stage T1 patients when comparing pre-surgery to post-surgery samples and four DEtRFs were found when comparing pre-surgery samples to NCPs. By performing a blast search, we found that sequences of DEtRFs aligned with genomic sequences pertaining to processes relevant to cancer development, such as enhancers, regulatory elements and CpG islands. Our findings display a number of tRFs that may hold potential as biomarkers for the diagnosis and recurrence-free survival of BC.

SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated.

The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates error-free repair followed by error-prone repair. Here, we have employed a multi-omics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death. Our results show that expression of genes involved in error-free and error-prone repair were both induced shortly after DNA damage, thus, challenging the established perception that the expression of error-prone repair genes is delayed. By combining transcriptomics and a sub-proteomics approach termed signalomics, we found that the temporal segregation of error-free and error-prone repair is primarily regulated after transcription, supporting the current literature. Furthermore, the heterology index (i.e., the binding affinity of LexA to the SOS box) was correlated to the maximum increase in gene expression and not to the time of induction of SOS genes. Finally, quantification of metabolites revealed increasing pyrimidine pools as a late feature of the SOS response. Our results elucidate how the SOS response is coordinated, showing a rapid transcriptional response and temporal regulation of mutagenesis on the protein and metabolite levels.

Correction: Gravina et al. ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma. Cancers 2022, 14, 289.

In the original publication [...].

ATX-101, a cell-penetrating protein targeting PCNA, can be safely administered as intravenous infusion in patients and shows clinical activity in a Phase 1 study.

Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved protein essential for DNA replication, repair and scaffold functions in the cytosol. Specific inhibition of PCNA in cancer cells is an attractive anti-cancer strategy. ATX-101 is a first-in-class drug targeting PCNA, primarily in cellular stress regulation. Multiple in vivo and in vitro investigations demonstrated anti-cancer activity of ATX-101 in many tumor types and a potentiating effect on the activity of anti-cancer therapies. Healthy cells were less affected. Based on preclinical data, a clinical phase 1 study was initiated. Twenty-five patients with progressive, late-stage solid tumors were treated with weekly ATX-101 infusions at four dose levels (20, 30, 45, 60 mg/m2). ATX-101 showed a favorable safety profile supporting that vital cellular functions are not compromised in healthy cells. Mild and moderate infusion-related reactions were observed in 64% of patients. ATX-101 was quickly cleared from blood with elimination half-lives of less than 30 min at all dose levels, probably due to both, a quick cell penetration and peptide digestion in serum, as demonstrated in vivo. No tumor responses were observed but stable disease was seen in 70% of the efficacy population (n = 20). Further studies have been initiated to provide evidence of efficacy. Trial registration numbers: ANZCTR 375262 and ANZCTR 375319.

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.

Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates.

The past few decades have been plagued by an increasing number of infections caused by antibiotic resistant bacteria. To mitigate the rise in untreatable infections, we need new antibiotics with novel targets and drug combinations that reduce resistance development. The novel ß-clamp targeting antimicrobial peptide BTP-001 was recently shown to have a strong additive effect in combination with the halogenated pyrrolopyrimidine JK-274. In this study, the molecular basis for this effect was examined by a comprehensive proteomic and metabolomic study of the individual and combined effects on Staphylococcus aureus. We found that JK-274 reduced activation of several TCA cycle enzymes, likely via increasing the cellular nitric oxide stress, and BTP-001 induced oxidative stress in addition to inhibiting replication, translation, and DNA repair processes. Analysis indicated that several proteins linked to stress were only activated in the combination and not in the single treatments. These results suggest that the strong additive effect is due to the activation of multiple stress responses that can only be triggered by the combined effect of the individual mechanisms. Importantly, the combination dose required to eradicate S. aureus was well tolerated and did not affect cell viability of immortalized human keratinocyte cells, suggesting a species-specific response. Our findings demonstrate the potential of JK-274 and BTP-001 as antibiotic drug candidates and warrant further studies.

Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA.

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.

X-ray repair cross complementing protein 1 in base excision repair.

X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma.

Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

Halogenated Pyrrolopyrimidines with Low MIC on Staphylococcus aureus and Synergistic Effects with an Antimicrobial Peptide.

Currently, there is a world-wide rise in antibiotic resistance causing burdens to individuals and public healthcare systems. At the same time drug development is lagging behind. Therefore, finding new ways of treating bacterial infections either by identifying new agents or combinations of drugs is of utmost importance. Additionally, if combination therapy is based on agents with different modes of action, resistance is less likely to develop. The synthesis of 21 fused pyrimidines and a structure-activity relationship study identified two 6-aryl-7H-pyrrolo [2,3-d] pyrimidin-4-amines with potent activity towards Staphylococcus aureus. The MIC-value was found to be highly dependent on a bromo or iodo substitution in the 4-benzylamine group and a hydroxyl in the meta or para position of the 6-aryl unit. The most active bromo and iodo derivatives had MIC of 8 mg/L. Interestingly, the most potent compounds experienced a four-fold lower MIC-value when they were combined with the antimicrobial peptide betatide giving MIC of 1-2 mg/L. The front runner bromo derivative also has a low activity towards 50 human kinases, including thymidylate monophosphate kinase, a putative antibacterial target.