bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒11‒22
27 papers selected by
Sean Rudd
Karolinska Institutet

  1. PLoS Genet. 2020 Nov 17. 16(11): e1009117
      Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.
  2. Mol Cancer Ther. 2020 Nov 17. pii: molcanther.0480.2020. [Epub ahead of print]
      Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival and chemotherapy resistance. While normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays and drug-target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's anti-proliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis addicted cancers.
  3. Nat Commun. 2020 11 17. 11(1): 5863
      Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.
  4. Nucleic Acids Res. 2020 Nov 19. pii: gkaa1048. [Epub ahead of print]
      Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
  5. Proc Natl Acad Sci U S A. 2020 Nov 17. pii: 202009506. [Epub ahead of print]
      Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition-G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.
    Keywords:  aneuploidy; cell cycle; chemotherapy resistance; cisplatin; paclitaxel
  6. Proc Natl Acad Sci U S A. 2020 Nov 17. pii: 202017637. [Epub ahead of print]
      The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.
    Keywords:  DNA polymerase; DNA polymerase δ; DNA replication; PCNA; sliding clamp
  7. EMBO J. 2020 Nov 19. e103654
      Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5' DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA-binding ability of CST is required for blocking MRE11-mediated nascent-strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent-strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.
    Keywords:  CST complex; DNA degradation; genome stability; nascent strand; replication stress
  8. Mol Cancer Res. 2020 Nov 18. pii: molcanres.0289.2020. [Epub ahead of print]
      Many chemotherapeutic drugs produce double-strand breaks (DSBs) on cancer cell DNA, thereby inducing cell death. However, the DNA damage response (DDR) enables cancer cells to overcome DNA damage and escape cell death, often leading to therapeutic resistance and unsuccessful outcomes. It is therefore important to develop inhibitors that target DDR proteins to render cancer cells hypersensitive to DNA damage. Here, we investigated the applicability of PFI-3, a recently developed bromodomain (BRD) inhibitor specifically targeting the SWI/SNF chromatin remodeler that functions to promote DSB repair, in cancer treatment. We verified that PFI-3 effectively blocks chromatin binding of its target BRDs and dissociates the corresponding SWI/SNF proteins from chromatin. We then found that, while having little toxicity as a single agent, PFI-3 synergistically sensitizes several human cancer cell lines to DNA damage induced by chemotherapeutic drugs such as doxorubicin. This PFI-3 activity occurs only for the cancer cells that require SWI/SNF for DNA repair. Our mechanism studies show that PFI-3 exerts the DNA damage-sensitizing effect by directly blocking SWI/SNF's chromatin binding, which leads to defects in DSB repair and aberrations in damage checkpoints, eventually resulting in increase of cell death primarily via necrosis and senescence. This work therefore demonstrates the activity of PFI-3 to sensitize cancer cells to DNA damage and its mechanism of action via SWI/SNF targeting, providing an experimental rationale for developing PFI-3 as a sensitizing agent in cancer chemotherapy. Implications: This study, revealing the activity of PFI-3 to sensitize cancer cells to chemotherapeutic drugs, provides an experimental rationale for developing this BRD inhibitor as a sensitizing agent in cancer chemotherapy.
  9. Sci Rep. 2020 11 16. 10(1): 19907
      Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin's effects. Amino acid deprivation enhanced cisplatin's cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin's biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug's side effects, and allow its use in cisplatin-resistant tumors.
  10. Trends Cancer. 2020 Nov 14. pii: S2405-8033(20)30284-3. [Epub ahead of print]
      DNA replication stress describes a state of impaired replication fork progress that triggers a cellular stress response to maintain genome stability and complete DNA synthesis. Replication stress is a common state that must be tolerated in many cancers. One promising therapeutic approach is targeting replication stress response factors such as the ataxia telangiectasia and rad 3-related kinase (ATR) or checkpoint kinase 1 (CHK1) kinases that some cancers depend upon to survive endogenous replication stress. However, research revealing the complexity of the replication stress response suggests new genetic interactions and candidate therapeutic targets. Many of these candidates regulate DNA transactions around reversed replication forks, including helicases, nucleases and alternative polymerases that promote fork stability and restart. Here we review emerging strategies to exploit replication stress for cancer therapy.
    Keywords:  DNA repair; DNA replication stress; chemotherapy; fork reversal; synthetic lethality
  11. Proc Natl Acad Sci U S A. 2020 Nov 16. pii: 202020189. [Epub ahead of print]
      Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.
    Keywords:  DNA replication; Pif1; replication bypass; replisome; single-molecule
  12. DNA Repair (Amst). 2020 Nov 02. pii: S1568-7864(20)30267-6. [Epub ahead of print]97 103007
      RecQL5, a mammalian RecQ family protein, is involved in the regulation of transcription elongation, DNA damage response, and DNA replication. Here, we identified and characterized an alternative splicing isoform of RECQL5 (RECQL5β1), which contains 17 additional amino acid residues within the RECQL5 KIX domain when compared with the canonical isoform (RECQL5β). RECQL5β1 had a markedly decreased binding affinity to RNA polymerase II (Pol II) and poorly competed with the transcription elongation factor TFIIS for binding to Pol II. As a result, this isoform has a weaker activity for repression of transcription elongation. In contrast, we discovered that RECQL5β1 could bind stronger to MRE11, which is a primary sensor of DNA double-strand breaks (DSBs). Furthermore, we found that RECQL5β1 promoted DNA repair in the RECQL5β1 rescue cells. These results suggest that RECQL5β mainly functions as a transcription repressor, while the newly discovered RECQL5β1 has a specialized role in DNA damage response. Taken together, our data suggest a cellular-functional specialization for each KIX splicing isoform in the cell.
    Keywords:  DNA damage response; MRE11; RECQL5; RNA polymerase II; Splicing isoform; Transcription regulation
  13. Oncogene. 2020 Nov 15.
      Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.
  14. EMBO J. 2020 Nov 20. e104400
      The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.
    Keywords:  ATM; DNA damage response; PIKKs; ataxia-telangiectasia; phosphoproteomics
  15. Nat Chem Biol. 2020 Nov 16.
      The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.
  16. NAR Cancer. 2020 Dec;2(4): zcaa033
      Identifying the mechanisms mediating cisplatin response is essential for improving patient response. Previous research has identified base excision repair (BER) and mismatch repair (MMR) activity in sensitizing cells to cisplatin. Cisplatin forms DNA adducts including interstrand cross-links (ICLs) that distort the DNA helix, forcing adjacent cytosines to become extrahelical. These extrahelical cytosines provide a substrate for cytosine deaminases. Herein, we show that APOBEC3 (A3) enzymes are capable of deaminating the extrahelical cytosines to uracils and sensitizing breast cancer cells to cisplatin. Knockdown of A3s results in resistance to cisplatin and induction of A3 expression in cells with low A3 expression increases sensitivity to cisplatin. We show that the actions of A3s are epistatic with BER and MMR. We propose that A3-induced cytosine deamination to uracil at cisplatin ICLs results in repair of uracils by BER, which blocks ICL DNA repair and enhances cisplatin efficacy and improves breast cancer outcomes.
  17. Nat Commun. 2020 11 17. 11(1): 5834
      Recent studies demonstrated a dramatically increased risk of leukemia in patients with a rare genetic disorder, Xeroderma Pigmentosum group C (XP-C), characterized by constitutive deficiency of global genome nucleotide excision repair (GG-NER). The genetic mechanisms of non-skin cancers in XP-C patients remain unexplored. In this study, we analyze a unique collection of internal XP-C tumor genomes including 6 leukemias and 2 sarcomas. We observe a specific mutational pattern and an average of 25-fold increase of mutation rates in XP-C versus sporadic leukemia which we presume leads to its elevated incidence and early appearance. We describe a strong mutational asymmetry with respect to transcription and the direction of replication in XP-C tumors suggesting association of mutagenesis with bulky purine DNA lesions of probably endogenous origin. These findings suggest existence of a balance between formation and repair of bulky DNA lesions by GG-NER in human body cells which is disrupted in XP-C patients.
  18. DNA Repair (Amst). 2020 Nov 08. pii: S1568-7864(20)30279-2. [Epub ahead of print]97 103019
      DNA double-strand breaks (DSBs) constitute one of the most cytotoxic forms of DNA damage and pose a significant threat to cell viability, survival, and homeostasis. DSBs have the potential to promote aneuploidy, cell death and potentially deleterious mutations that promote tumorigenesis. Homologous recombination (HR) is one of the main DSB repair pathways and while being essential for cell survival under genotoxic stress, it requires proper regulation to avoid chromosome rearrangements. Here, we characterize the Saccharomyces cerevisiae E3 ubiquitin ligase/putative helicase Irc20 as a regulator of HR. Using purified Irc20, we show that it can hydrolyze ATP in the presence and absence of DNA, but does not increase access to DNA within a nucleosome. In addition, we show that both the ATPase and ubiquitin ligase activities of Irc20 are required for suppressing the spontaneous formation of recombination foci. Finally, we demonstrate a role for Irc20 in promoting Rad51 chromatin association and the removal of Rad52 recombinase from chromatin, thus facilitating subsequent HR steps and directing recombination to more error-free modes.
    Keywords:  DNA repair; Homologous recombination; Irc20; SUMO; Ubiquitin ligase
  19. iScience. 2020 Oct 23. 23(10): 101604
      SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.
    Keywords:  Cancer; Cell Biology; Molecular Biology
  20. J Mol Biol. 2020 Nov 17. pii: S0022-2836(20)30633-1. [Epub ahead of print]
      There are two major pathways for repairing DNA double-strand breaks (DSBs): homologous directed recombination (HDR) and non-homologous end-joining (NHEJ). While NHEJ functions throughout the cell cycle, HDR is only possible during S/G2 phases, suggesting that there are cell cycle-specific mechanisms regulating the balance between the two repair systems. The regulation exerted by CDKs on HDR has been extensively demonstrated, and here we present evidence that the CDK Pho85, in association with the G1 cyclin Pcl1, phosphorylates Yku80 on Ser 623 to regulate NHEJ activity. Cells bearing a non-phosphorylatable version of Yku80 show increased NHEJ and reduced HDR activity. Accordingly, yku80S623A cells present diminished viability upon treatment with the DSB-producer bleomycin, specifically in the G2 phase of the cell cycle. Interestingly, the mutation of the equivalent residue in human Ku80 increases sensitivity to bleomycin in several cancer cell lines, suggesting that this mechanism is conserved in humans. Altogether, our results reveal a new mechanism whereby G1-CDKs mediate the choice between HDR and NHEJ repair pathways, putting the error prone NHEJ on a leash and enabling error free HDR in G2 when homologous sequences are available.
    Keywords:  DNA repair; HDR; Ku80; NHEJ; Pho85
  21. Nat Rev Mol Cell Biol. 2020 Nov 18.
      The journey of RNA polymerase II (Pol II) as it transcribes a gene is anything but a smooth ride. Transcript elongation is discontinuous and can be perturbed by intrinsic regulatory barriers, such as promoter-proximal pausing, nucleosomes, RNA secondary structures and the underlying DNA sequence. More substantial blocking of Pol II translocation can be caused by other physiological circumstances and extrinsic obstacles, including other transcribing polymerases, the replication machinery and several types of DNA damage, such as bulky lesions and DNA double-strand breaks. Although numerous different obstacles cause Pol II stalling or arrest, the cell somehow distinguishes between them and invokes different mechanisms to resolve each roadblock. Resolution of Pol II blocking can be as straightforward as temporary backtracking and transcription elongation factor S-II (TFIIS)-dependent RNA cleavage, or as drastic as premature transcription termination or degradation of polyubiquitylated Pol II and its associated nascent RNA. In this Review, we discuss the current knowledge of how these different Pol II stalling contexts are distinguished by the cell, how they overlap with each other, how they are resolved and how, when unresolved, they can cause genome instability.
  22. Biochem Soc Trans. 2020 Nov 16. pii: BST20200678. [Epub ahead of print]
      Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.
    Keywords:  DNA polymerases; DNA replication and recommbination; PCNA; mutagenesis; proten-protein interaction; translesion DNA synthesis
  23. J Am Chem Soc. 2020 Nov 17.
      The DNA glycosylase MutY prevents deleterious mutations resulting from guanine oxidation by recognition and removal of adenine (A) misincorporated opposite 8-oxo-7,8-dihydroguanine (OG). Correct identification of OG:A is crucial to prevent improper and detrimental MutY-mediatedadenine excision from G:A or T:A base pairs. Here we present a structure-activity relationship (SAR) study using analogues of A to probe the basis for OG:A specificity of MutY. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency. These data show that H-bonding and electrostatic interactions of the base within the MutY active site modulate the lability of the N-glycosidic bond. A analogues that were not excised from duplex DNA as efficiently as predicted by calculations provided insight into other required structural features, such as steric fit and H-bonding within the active site for proper alignment with MutY catalytic residues. We also determined MutY-mediated repair of A analogues paired with OG within the context of a DNA plasmid in bacteria. Remarkably, the magnitudes of decreased in vitro MutY excision rates with different A analogue duplexes do not correlate with the impact on overall MutY-mediated repair. The feature that most strongly correlated with facile cellular repair was the ability of the A analogues to H-bond with the Hoogsteen face of OG. Notably, base pairing of A with OG uniquely positions the 2-amino group of OG in the major groove and provides a means to indirectly select only these inappropriately placed adenines for excision. This highlights the importance of OG lesion detection for efficient MutY-mediated cellular repair. The A analogue SARs also highlight the types of modifications tolerated by MutY and will guide the development of specific probes and inhibitors of MutY.
  24. Carcinogenesis. 2020 Nov 18. pii: bgaa119. [Epub ahead of print]
      Deregulation of MYC occurs in a broad range of human cancers and often predicts poor prognosis and resistance to therapy. However, directly targeting oncogenic MYC remains unsuccessful, and indirectly inhibiting MYC emerges as a promising approach. Checkpoint kinase 1 (CHK1) is a protein kinase that coordinates the G2/M cell cycle checkpoint and protects cancer cells from excessive replicative stress. Using c-MYC-mediated T-cell acute lymphoblastic leukemia (T-ALL) and N-MYC-driven neuroblastoma as model systems, we reveal that both c-MYC and N-MYC directly bind to the CHK1 locus and activate its transcription. CHIR-124, a selective CHK1 inhibitor, impairs cell viability and induces remarkable synergistic lethality with mTOR inhibitor rapamycin in MYC-overexpressing cells. Mechanistically, rapamycin inactivates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD), the essential enzyme for the first three steps of de novo pyrimidine synthesis, and deteriorates CHIR-124-induced replicative stress. We further demonstrate that dual treatments impede T-ALL and neuroblastoma progression in vivo. These results suggest simultaneous targeting of CHK1 and mTOR as a novel and powerful co-treatment modality for MYC-mediated tumors.
  25. Anal Chim Acta. 2020 Dec 01. pii: S0003-2670(20)30939-9. [Epub ahead of print]1139 119-128
      Several novel non-typical nucleoside analogs were examined as potential fluorescent indicators of purine-nucleoside phosphorylase (PNP) activity in human blood. The substrates included N7-riboside of 8-aza-2,6-diaminopurine, N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine. Reaction rates and apparent Michaelis' constants were determined in 1000-fold blood lysates and compared with those for reference compounds, guanosine and 7-methylguanosine. It was concluded that the most promising for assaying human PNP in biological material was N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine was optimal for the E. coli PNP, both offering at least 10-fold improvement in sensitivity relative to conventional assays. Other potential applications of this approach are discussed.
    Keywords:  Blood activity; Fluorescent/fluorogenic substrates; Nucleoside and nucleobase analogs; Purine-nucleoside phosphorylase
  26. Food Chem Toxicol. 2020 Nov 17. pii: S0278-6915(20)30755-9. [Epub ahead of print] 111865
      The enzyme-modified comet assay was developed in order to detect DNA lesions other than those detected by the standard version (single and double strand breaks and alkali-labile sites). Various lesion-specific enzymes, from the DNA repair machinery of bacteria and humans, have been combined with the comet assay, allowing detection of different oxidized and alkylated bases as well as cyclobutane pyrimidine dimers, mis-incorporated uracil and apurinic/apyrimidinic sites. The enzyme-modified comet assay has been applied in different fields - human biomonitoring, environmental toxicology, and genotoxicity testing (both in in vitro and in in vivo) - as well as in basic research. Up to now, twelve enzymes have been employed; here we describe the enzymes and give examples of studies in which they have been applied. The bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII) have been extensively used while others have been used only rarely. Adding further enzymes to the comet assay toolbox could potentially increase the variety of DNA lesions that can be detected. The enzyme-modified comet assay can play a crucial role in the elucidation of the mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context. mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context.
    Keywords:  AP-sites; alkaline comet assay; alkylated lesions; cyclobutane pyrimidine dimers; oxidized lesions; uracil mis-incorporation
  27. J Biol Chem. 2020 11 16. pii: jbc.REV120.011985. [Epub ahead of print]
      Protein synthesis is an energetically costly cellular activity. It is therefore important that the process of mRNA translation remains in excellent synchrony with cellular metabolism and its energy reserves. Unregulated translation could lead to the production of incomplete, mistranslated, or misfolded proteins, squandering the energy needed for cellular sustenance, and causing cytotoxicity. One-carbon metabolism (OCM), an integral part of cellular intermediary metabolism, produces a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl). These OCM intermediates are required for the production of amino acids like methionine, and biomolecules such as purines, thymidylate, and redox regulators. In this review, we discuss how OCM impacts the translation apparatus (composed of ribosome, tRNA, mRNA, and translation factors) and regulates crucial steps in protein synthesis. More specifically, we address how the OCM metabolites regulate the fidelity and rate of translation initiation in bacteria and eukaryotic organelles such as mitochondria. Modulation of the fidelity of translation initiation by OCM opens new avenues to understand alternative translation mechanisms involved in stress tolerance and drug resistance.
    Keywords:  3GC base pairs; RNA; RNA methylation; RNA methyltransferase; RNA modification; S-adenosylmethionine (SAM); folate; formylation; initiator tRNA; methylations; mitochondria; one-carbon metabolism; ribosome; ribosome heterogeneity