bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–03–20
29 papers selected by
Sean Rudd, Karolinska Institutet
  1. Global genomic instability caused by reduced expression of DNA polymerase ε in yeast.
  2. Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress.
  3. Ribonucleotide Incorporation by Eukaryotic B-family Replicases and Its Implications for Genome Stability.
  4. New insights into the DNA repair pathway choice with NuA4/TIP60.
  5. Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy.
  6. In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing.
  7. A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins.
  8. Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells.
  9. Characterization of DNA-PK-Bound End Fragments Using GLASS-ChIP.
  10. DNA-Protein Crosslinks and Their Resolution.
  11. Approaches to Monitor Termination of DNA Replication Using Xenopus Egg Extracts.
  12. The nuclease activity of DNA2 promotes Exonuclease 1-independent mismatch repair.
  13. Combinatorial targeting of Hippo-STRIPAK and PARP elicits synthetic lethality in gastrointestinal cancers.
  14. FMRP promotes transcription-coupled homologous recombination via facilitating TET1-mediated m5C RNA modification demethylation.
  15. Cracking the homologous recombination deficiency code: how to identify responders to PARP inhibitors.
  16. Editorial: Genome Instability: Old Problem, New Solutions.
  17. A Novel Synthetic Lethal Approach to Target MYC-Driven Cancers.
  18. Pairing structural reconstruction with catalytic competence to evaluate the mechanisms of key enzymes in the folate-mediated one-carbon pathway.
  19. Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability.
  20. High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.
  21. Rational Design of an Orally Active Anticancer Fluoropyrimidine, Pencitabine, a Hybrid of Capecitabine and Gemcitabine.
  22. One residue-one function.
  23. Acetylation of H3K56 orchestrates UV-responsive chromatin events that generate DNA accessibility during Nucleotide Excision Repair.
  24. Epigenetic anti-cancer treatment with a stabilized carbocyclic Decitabine analogue.
  25. Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of 13C-Bicarbonate Incorporation.
  26. Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells.
  27. The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.
  28. Targeted Formation of 8-Oxoguanine in Telomeres.
  29. ATR and CDK4/6 inhibition target the growth of methotrexate-resistant choriocarcinoma.
  1. Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2119588119
    Global genomic instability caused by reduced expression of DNA polymerase ε in yeast.
    Ke Zhang, Yang Sui, Wu-Long Li, Gen Chen, Xue-Chang Wu, Robert J Kokoska, Thomas D Petes, Dao-Qiong Zheng.
      SignificanceAlthough most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.
    Keywords:  DNA polymerase; DNA replication stress; genome instability; loss of heterozygosity; mitotic recombination
    DOI:  https://doi.org/10.1073/pnas.2119588119
  2. Life Sci Alliance. 2022 Jun;pii: e202101249. [Epub ahead of print]5(6):
    Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress.
    Yoshitami Hashimoto, Hirofumi Tanaka.
      Mre11 is a versatile exo-/endonuclease involved in multiple aspects of DNA replication and repair, such as DSB end processing and checkpoint activation. We previously demonstrated that forced mitotic entry drives replisome disassembly at stalled replication forks in Xenopus egg extracts. Here, we examined the effects of various chemical inhibitors using this system and discovered a novel role of Mre11 exonuclease activity in promoting mitotic entry under replication stress. Mre11 activity was necessary for the initial progression of mitotic entry in the presence of stalled forks but unnecessary in the absence of stalled forks or after mitotic entry. In the absence of Mre11 activity, mitotic CDK was inactivated by Wee1/Myt1-dependent phosphorylation, causing mitotic exit. An inhibitor of Wee1/Myt1 or a nonphosphorylatable CDK1 mutant was able to partially bypass the requirement of Mre11 for mitotic entry. These results suggest that Mre11 exonuclease activity facilitates the processing of stalled replication forks upon mitotic entry, which attenuates the inhibitory pathways of mitotic CDK activation, leading to irreversible mitotic progression and replisome disassembly.
    DOI:  https://doi.org/10.26508/lsa.202101249
  3. Annu Rev Biochem. 2022 Feb 14.
    Ribonucleotide Incorporation by Eukaryotic B-family Replicases and Its Implications for Genome Stability.
    Jessica S Williams, Thomas A Kunkel.
      Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-032620-110354
  4. DNA Repair (Amst). 2022 Mar 03. pii: S1568-7864(22)00044-1. [Epub ahead of print]113 103315
    New insights into the DNA repair pathway choice with NuA4/TIP60.
    Anahita Lashgari, Pata-Eting Kougnassoukou Tchara, Jean-Philippe Lambert, Jacques Côté.
      In eukaryotic cells, DNA double-strand breaks (DSBs) can be repaired through two main pathways, non-homologous end-joining (NHEJ) or homologous recombination (HR). The selection of the repair pathway choice is governed by an antagonistic relationship between repair factors specific to each pathway, in a cell cycle-dependent manner. The molecular mechanisms of this decision implicate post-translational modifications of chromatin surrounding the break. Here, we discuss the recent advances regarding the function of the NuA4/TIP60 histone acetyltransferase/chromatin remodeling complex during DSBs repair. In particular, we emphasise the contribution of NuA4/TIP60 in repair pathway choice, in collaboration with the SAGA acetyltransferase complex, and how they regulate chromatin dynamics, modify non-histone substrates to allow DNA end resection and recombination.
    Keywords:  Chromatin remodeling; Double-strand break; Histone modifications; NuA4; SAGA; TIP60
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103315
  5. Methods Mol Biol. 2022 ;2444 81-103
    Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy.
    Jessica Jackson, Alessandro Vindigni.
      Single-stranded DNA gaps are frequent structures that accumulate on newly synthesized DNA under conditions of replication stress. The identification of these single-stranded DNA gaps has been instrumental to uncover the mechanisms that allow the DNA replication machinery to skip intrinsic replication obstacles or DNA lesions. DNA fiber assays provide an essential tool for detecting perturbations in DNA replication fork dynamics genome-wide at single molecule resolution along with identifying the presence of single-stranded gaps when used in combination with S1 nuclease. However, electron microscopy is the only technique allowing the actual visualization and localization of single-stranded DNA gaps on replication forks. This chapter provides a detailed method for visualizing single-stranded DNA gaps at the replication fork by electron microscopy including psoralen cross-linking of cultured mammalian cells, extraction of genomic DNA, and finally enrichment of replication intermediates followed by spreading and platinum rotary shadowing of the DNA onto grids. Discussion on identification and analysis of these gaps as well as on the advantages and disadvantages of electron microscopy relative to the DNA fiber technique is also included.
    Keywords:  DNA replication; DNA replication stress; Electron microscopy; Replication structures; ssDNA gaps
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_6
  6. Methods Mol Biol. 2022 ;2444 207-225
    In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing.
    Meiling Wang, Cody M Rogers, Dauren Alimbetov, Weixing Zhao.
      RAD51-mediated homologous recombination (HR) is a conserved mechanism for the repair of DNA double-strand breaks and the maintenance of DNA replication forks. Several breast and ovarian tumor suppressors, including BRCA1 and BARD1, have been implicated in HR since their discovery in the 1990s. However, a holistic understanding of how they participate in HR has been hampered by the immense challenge of expressing and purifying these large and unstable protein complexes for mechanistic analysis. Recently, we have overcome such a challenge for the BRCA1-BARD1 complex, allowing us to demonstrate its pivotal role in HR via the promotion of RAD51-mediated DNA strand invasion. In this chapter, we describe detailed procedures for the expression and purification of the BRCA1-BARD1 complex and in vitro assays using this tumor suppressor complex to examine its ability to promote RAD51-mediated homologous DNA pairing. This includes two distinct biochemical assays, namely, D-loop formation and synaptic complex assembly. These methods are invaluable for studying the BRCA1-BARD1 complex and its functional interplay with other factors in the HR process.
    Keywords:  BRCA1-BARD1; D-loop formation; Homologous recombination; RAD51; Synaptic complex assembly
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_13
  7. Methods Mol Biol. 2022 ;2444 15-27
    A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins.
    Bo-Ruei Chen, Barry P Sleckman.
      DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.
    Keywords:  CRISPR/Cas9; DNA end resection; Genome-wide screen; HR; NHEJ; RPA
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_2
  8. Front Immunol. 2022 ;13 765284
    Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells.
    Felix Meyer, Anna Maria Engel, Ann Kristin Krause, Tim Wagner, Lena Poole, Anna Dubrovska, Claudia Peitzsch, Kai Rothkamm, Cordula Petersen, Kerstin Borgmann.
      Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p<0.0001). Therefore, disruption of HR-mediated processes, particularly in replication, opens a CSC-specific radiosensitization option by enhancing their intracellular immune response.
    Keywords:  ATR inhibition; DNA repair; breast cancer stem cells (BCSCs); cellular immuneresponse; homologous recombination; immunogenic cytosolic dsDNA; radioresistance; replication stress
    DOI:  https://doi.org/10.3389/fimmu.2022.765284
  9. Methods Mol Biol. 2022 ;2444 171-182
    Characterization of DNA-PK-Bound End Fragments Using GLASS-ChIP.
    Rajashree A Deshpande, Tanya T Paull.
      Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent protein kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.
    Keywords:  DNA repair; DNA-PK; Double-strand breaks; MRN complex
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_11
  10. Annu Rev Biochem. 2022 Feb 18.
    DNA-Protein Crosslinks and Their Resolution.
    Pedro Weickert, Julian Stingele.
      Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-032620-105820
  11. Methods Mol Biol. 2022 ;2444 105-123
    Approaches to Monitor Termination of DNA Replication Using Xenopus Egg Extracts.
    Tamar Kavlashvili, James M Dewar.
      DNA replication is crucial for cell viability and genome integrity. Despite its crucial role in genome duplication, the final stage of DNA replication, which is termed termination, is relatively unexplored. Our knowledge of termination is limited by cellular approaches to study DNA replication, which cannot readily detect termination. In contrast, the Xenopus laevis egg extract system allows for all of DNA replication to be readily detected. Here we describe the use of this system and assays to monitor replication termination.
    Keywords:  Chromatin capture; DNA replication; DNA synthesis; Decatenation; Fork merger; Ligation; Xenopus egg extracts
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_7
  12. J Biol Chem. 2022 Mar 14. pii: S0021-9258(22)00271-X. [Epub ahead of print] 101831
    The nuclease activity of DNA2 promotes Exonuclease 1-independent mismatch repair.
    Lyudmila Y Kadyrova, Basanta K Dahal, Vaibhavi Gujar, James M Daley, Patrick Sung, Farid A Kadyrov.
      The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of Exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.
    DOI:  https://doi.org/10.1016/j.jbc.2022.101831
  13. J Clin Invest. 2022 Mar 15. pii: e155468. [Epub ahead of print]
    Combinatorial targeting of Hippo-STRIPAK and PARP elicits synthetic lethality in gastrointestinal cancers.
    Liwei An, Zhifa Cao, Pingping Nie, Hui Zhang, Zhenzhu Tong, Fan Chen, Yang Tang, Yi Han, Wenjia Wang, Zhangting Zhao, Qingya Zhao, Yuqin Yang, Yuanzhi Xu, Gemin Fang, Lei Shi, Huixiong Xu, Haiqing Ma, Shi Jiao, Zhaocai Zhou.
      The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli to result in intracellular activities. Previously, we discovered STRIPAK to be a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex to be an essential player for the control of DNA double-strand break (DSB) repair and genomic stability. Specifically, the MST1/2 kinases were found, independent of the classical Hippo signaling, to directly phosphorylate ZMYND8 and hence result in suppression of DNA repair in the nucleus. In response to genotoxic stress, the cGAS-STING pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via a TBK1-induced structural stabilization of the SIKE1-SLMAP arm. As such, STRIPAK-mediated MST1/2 inactivation was found to increase the DSB repair capacity of cancer cells and to endow these cells with resistance to radio/chemotherapy and PARP inhibition. Importantly, targeting the STRIPAK assembly with each of three distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and re-sensitize cancer cells to PARPi in both animal and patient-derived tumor models. Overall, our findings not only uncovered a previously unrecognized role for STRIPAK in modulating DSB repair, but also provided translational implications of co-targeting STRIPAK and PARP for a new type of synthetic lethality anti-cancer therapy.
    Keywords:  Cell Biology; Drug therapy; Gastric cancer; Gastroenterology; Molecular biology
    DOI:  https://doi.org/10.1172/JCI155468
  14. Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2116251119
    FMRP promotes transcription-coupled homologous recombination via facilitating TET1-mediated m5C RNA modification demethylation.
    Haibo Yang, Yumin Wang, Yufei Xiang, Tribhuwan Yadav, Jian Ouyang, Laiyee Phoon, Xueping Zhu, Yi Shi, Lee Zou, Li Lan.
      SignificanceThis study shows that Fragile X mental retardation protein (FMRP) promotes messenger RNA (mRNA)-dependent recombination via facilitating ten-eleven translocation protein 1 (TET1)-mediated mRNA methyl-5-cytosine (m5C) demethylation. Loss of FMRP leads to damage induced mRNA m5C and R-loop accumulation at sites of active transcription, defective recombination repair, and increased radiosensitivity of tumor cells. FMRP-dependent RNA m5C demethylation and R-loop resolving during DNA repair are important for repair completion and the maintenance of genome stability. The removal of m5C by the FMRP-TET1 axis is coupled with R-loop dissolution, which ensures proper completion of DNA repair and survival of cells after DNA damage. These findings significantly advance our understanding of the regulation of RNA modifications in R-loop dynamics during DNA repair.
    Keywords:  DNA damage repair; FMRP; TRDMT1; m5C; mRNA modification
    DOI:  https://doi.org/10.1073/pnas.2116251119
  15. Eur J Cancer. 2022 Mar 10. pii: S0959-8049(22)00070-3. [Epub ahead of print]166 87-99
    Cracking the homologous recombination deficiency code: how to identify responders to PARP inhibitors.
    Lola Paulet, Alexis Trecourt, Alexandra Leary, Julien Peron, Françoise Descotes, Mojgan Devouassoux-Shisheboran, Karen Leroy, Benoit You, Jonathan Lopez.
      DNA double-strand breaks are the most critical DNA damage to cells, and their repair is tightly regulated to maintain cellular integrity. Some cancers exhibit homologous recombination deficiency (HRD), a faithful double-strand break repair system, making them more sensitive to poly (ADP ribose) polymerase inhibitors (PARPi). PARPi have shown substantial efficacy in BRCA-mutated ovarian cancer for several years, and their indication has gradually been extended to other tumour locations such as breast, prostate and pancreas. More recently, PARPi were demonstrated to be effective in cancers with an HRD phenotype beyond BRCA mutations. Today, a major challenge is developing tests capable of detecting the HRD phenotype of cancers (HRD tests) and predicting sensitivity to PARPi to select patients likely to benefit from this therapy. This review provides a synthesis of the existing HRD tests, divided into three main approaches to detect HRD: the investigation of the HRD causes, the study of its consequences and the evaluation of the HR activity itself.
    Keywords:  BRCA; Genomic scars; HRD tests; Homologous recombination deficiency; PARP inhibitors; RAD51 foci
    DOI:  https://doi.org/10.1016/j.ejca.2022.01.037
  16. Front Cell Dev Biol. 2022 ;10 868038
    Editorial: Genome Instability: Old Problem, New Solutions.
    Marta Popovic, Vivian Kahl, Nicolas C Hoch.
      
    Keywords:  DNA Damage; DNA Repair; DNA Replication; Emerging methods; Genomic Instability
    DOI:  https://doi.org/10.3389/fcell.2022.868038
  17. Cancer Res. 2022 Mar 15. 82(6): 969-971
    A Novel Synthetic Lethal Approach to Target MYC-Driven Cancers.
    Roman M Chabanon, Sophie Postel-Vinay.
      The MYC proto-oncogene family encompasses three related transcription factors (MYC, MYCL, and MYCN), which are master regulators of cellular programs orchestrating multiple hallmarks of cancer, including proliferation, metabolism, invasiveness, and immune surveillance. MYC activation is one of the most frequent alterations in cancer, induced by genetic, epigenetic, or posttranslational alterations of MYC itself, or of MYC-related proteins or pathways. Sun and colleagues found a unique function of the rate-limiting nucleotide synthesis enzyme CTP synthase 1 (CTPS1) in the survival of MYC-driven cancer cells. They further identified a novel synthetic lethal strategy to combat MYC-driven cancers by combining CTPS1 inhibitors with ataxia telangiectasia and Rad3-related protein inhibitors, which exploits the inherent vulnerability of MYC-driven tumors to nucleotide shortage and DNA replication stress. These findings open novel therapeutic avenues for targeting the traditionally "undruggable" MYC-driven cancers, which represent one of the highest unmet clinical needs in cancer. See related article by Sun et al. p. 1013.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-0302
  18. FEBS J. 2022 Mar 18.
    Pairing structural reconstruction with catalytic competence to evaluate the mechanisms of key enzymes in the folate-mediated one-carbon pathway.
    Li Na Zhao, Philipp Kaldis.
      Mammalian metabolism comprises a series of interlinking pathways that include two major cycles: the folate and methionine cycles. The folate-mediated metabolic cycle uses several oxidation states of tetrahydrofolate to carry activated one-carbon units to be readily used and interconverted within the cell, which are required for nucleotide synthesis, methylation and metabolism, particularly for proliferation of cancer cells. Based on the latest progress in genome-wide CRISPR loss-of-function viability screening of 789 cell lines, we focus on the most cancer dependent enzymes in this pathway, especially those that are hyperactivated in cancer, to provide new insight into the chemical basis for cancer drug development. Since the complete 3D structure of several of these enzymes in their active form are not available, we used homology modeling integrated with the interpretation of the reaction mechanism, and have reconstructed the most likely scenario for the reactions to take place paired with their catalytic cycle that provides a testable framework for this pathway.
    Keywords:  DHFR; Enzyme catalysis; Enzyme reaction mechanism; Folate; Folate metabolism; Folate-mediated one-carbon pathway; MTHFD1; MTHFD2; MTHFD2L; SHMT2; TYMS
    DOI:  https://doi.org/10.1111/febs.16439
  19. Microb Cell. 2022 Mar 07. 9(3): 52-68
    Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability.
    Alexandra Berroyer, Albino Bacolla, John A Tainer, Nayun Kim.
      Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic increase in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.
    Keywords:  G-quadruplex; Topoisomerase; genome instability; recombination; transcription
    DOI:  https://doi.org/10.15698/mic2022.03.771
  20. Elife. 2022 Mar 15. pii: e73943. [Epub ahead of print]11
    High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.
    Mingrui Duan, Smitha Sivapragasam, Jacob S Antony, Jenna Ulibarri, John M Hinz, Gregory M K Poon, John J Wyrick, Peng Mao.
      DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of UV damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.
    Keywords:  S. cerevisiae; cancer biology; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.73943
  21. ACS Med Chem Lett. 2022 Mar 10. 13(3): 409-416
    Rational Design of an Orally Active Anticancer Fluoropyrimidine, Pencitabine, a Hybrid of Capecitabine and Gemcitabine.
    Thomas I Kalman.
      The structure of the anticancer drug capecitabine was redesigned to prevent metabolic conversion to 5-fluorouracil and its associated potentially fatal toxicities. The resulting cytidine analogue, pencitabine, is a hybrid of capecitabine and gemcitabine, another anticancer drug in clinical use. Preliminary biological evaluation revealed that pencitabine is cytotoxic in vitro in cell culture and orally active in vivo in a human xenograft test system. Pencitabine may mimic the known therapeutically advantageous combination of its parent drugs. Pencitabine is postulated to interfere with DNA synthesis and function by inhibiting multiple nucleotide-metabolizing enzymes and by misincorporation into DNA. Based on detailed mechanistic analyses and literature precedents, the hypothesis is put forward that the significant DNA damage caused by pencitabine may be accounted for by two additional effects not shown by the parent drugs: inhibition of DNA glycosylases involved in base excision repair and of DNA (cytosine-5)-methyltransferase involved in epigenetic regulation of cellular metabolism.
    DOI:  https://doi.org/10.1021/acsmedchemlett.1c00565
  22. Science. 2022 Mar 18. 375(6586): 1232-1233
    One residue-one function.
    Frédéric Berger.
      The histone H3.1 variant deposited at replication forks docks DNA repair machinery.
    DOI:  https://doi.org/10.1126/science.abo4219
  23. DNA Repair (Amst). 2022 Mar 09. pii: S1568-7864(22)00046-5. [Epub ahead of print]113 103317
    Acetylation of H3K56 orchestrates UV-responsive chromatin events that generate DNA accessibility during Nucleotide Excision Repair.
    Preeti Khan, Ronita Nag Chaudhuri.
      Histone modifications have long been related to DNA damage response. Nucleotide excision repair pathway that removes helix-distorting lesions necessitates DNA accessibility through chromatin modifications. Previous studies have linked H3 tail residue acetylation to UV-induced NER. Here we present evidences that acetylation of H3K56 is crucial for early phases of NER. Using H3K56 mutants K56Q and K56R, which mimic acetylated and unacetylated lysines respectively, we show that recruitment of the repair factor Rad16, a Swi/Snf family member is dependent on H3K56 acetylation. With constitutive H3K56 acetylation, Rad16 recruitment became UV-independent. Furthermore, H3K56 acetylation promoted UV-induced hyperacetylation of H3K9 and H3K14. Importantly, constitutive H3K56 acetylation prominently increased chromatin accessibility. During NER, lack of H3K56 acetylation that effectively aborted H3 tail residue acetylation and Rad16 recruitment, thus failed to impart essential chromatin modulations. The NER-responsive oscillation of chromatin structure observed in wild type, was distinctly eliminated in absence of H3K56 acetylation. In vitro assay with wild type and H3K56 mutant cell extracts further indicated that absence of H3K56 acetylation negatively affected DNA relaxation during NER. Overall, H3K56 acetylation regulates Rad16 redistribution and UV-induced H3 tail residue hyperacetylation, and the resultant modification code promotes chromatin accessibility and recruitment of subsequent repair factors during NER.
    Keywords:  Chromatin modification; Chromatin regulation; DNA damage; H3K56; Histone acetylation; Nucleotide excision repair; Rad16
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103317
  24. Chemistry. 2022 Mar 14.
    Epigenetic anti-cancer treatment with a stabilized carbocyclic Decitabine analogue.
    Franziska R Traube, Natércia F Brás, Wynand P Roos, Corinna C Sommermann, Tamara Diehl, Robert J Mayer, Armin R Ofial, Markus Müller, Hendrik Zipse, Thomas Carell.
      5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.
    Keywords:  5-aza-2'-deoxycytidine; DNA damage; DNA hypomethylating agents; DNA-methyltransferases; acute myeloid leukemia
    DOI:  https://doi.org/10.1002/chem.202200640
  25. Biochemistry. 2022 Mar 14.
    Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of 13C-Bicarbonate Incorporation.
    Hyungjoo Shon, Elena A Matveeva, Ella C Jull, Yijia Hu, Tiffany A Coupet, Young-Sam Lee.
      Human phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimdiazole succinocarboxamide synthetase (PAICS) is a dual activity enzyme catalyzing two consecutive reactions in de novo purine nucleotide synthesis. Crystallographic structures of recombinant human PAICS suggested the channeling of 4-carboxy-5-aminoimidazole-1-ribose-5'-phosphate (CAIR) between two active sites of PAICS, while a prior work of an avian PAICS suggested otherwise. Here, we present time-course mass spectrometric data supporting the channeling of CAIR between domains of recombinant human PAICS. Time-course mass spectral analysis showed that CAIR added to the bulk solution (CAIRbulk) is decarboxylated and re-carboxylated before the accumulation of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR). An experiment with 13C-bicarbonate showed that SAICAR production was proportional to re-carboxylated CAIR instead of total CAIR or CAIRbulk. This result indicates that the SAICAR synthase domain selectively uses enzyme-made CAIR over CAIRbulk, which is consistent with the channeling model. This channeling between PAICS domains may be a part of a larger channeling process in de novo purine nucleotide synthesis.
    DOI:  https://doi.org/10.1021/acs.biochem.1c00803
  26. Front Mol Biosci. 2022 ;9 847829
    Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells.
    Patrycja Wińska, Łukasz Widło, Elżbieta Senkara, Mirosława Koronkiewicz, Jarosław M Cieśla, Alicja Krzyśko, Katarzyna Skierka, Joanna Cieśla.
      Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
    Keywords:  CX-4945; acute lymphoblastic leukemia cells CCRF-CEM; dihydrofolate reductase; protein kinase CK2; protein–protein interaction; serine hydroxymethyltransferase; thymidylate synthase
    DOI:  https://doi.org/10.3389/fmolb.2022.847829
  27. Nucleic Acids Res. 2022 Mar 14. pii: gkac172. [Epub ahead of print]
    The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.
    Suravi Pramanik, Yingling Chen, Heyu Song, Irine Khutsishvili, Luis A Marky, Sutapa Ray, Amarnath Natarajan, Pankaj K Singh, Kishor K Bhakat.
      Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.
    DOI:  https://doi.org/10.1093/nar/gkac172
  28. Methods Mol Biol. 2022 ;2444 141-159
    Targeted Formation of 8-Oxoguanine in Telomeres.
    Ryan P Barnes, Sanjana A Thosar, Elise Fouquerel, Patricia L Opresko.
      Mammalian telomeres are guanine-rich sequences which cap the ends of linear chromosomes. While recognized as sites sensitive to oxidative stress, studies on the consequences of oxidative damage to telomeres have been primarily limited to experimental conditions which cause oxidative damage throughout the whole genome and cell. We developed a chemoptogenetic tool (FAP-mCER-TRF1) to specifically induce singlet oxygen at telomeres, resulting in the formation of the common oxidative lesion 8-oxo-guanine. Here, we describe this tool and detail how to generate cell lines which express FAP-mCER-TRF1 at telomeres and verify the formation of 8-oxo-guanine.
    Keywords:  8-oxo-guanine; Chemoptogenetic tool; DNA damage; Oxidative stress; TRF1; Telomeres
    DOI:  https://doi.org/10.1007/978-1-0716-2063-2_9
  29. Oncogene. 2022 Mar 18.
    ATR and CDK4/6 inhibition target the growth of methotrexate-resistant choriocarcinoma.
    Marina Georgiou, Panagiota Ntavelou, William Stokes, Rajat Roy, Geoffrey J Maher, Tsvetana Stoilova, Callum P Rakhit, Miguel Martins, Paul Ajuh, Neil Horowitz, Ross S Berkowitz, Kevin Elias, Michael J Seckl, Olivier E Pardo.
      Low-risk gestational trophoblastic neoplasia including choriocarcinoma is often effectively treated with Methotrexate (MTX) as a first line therapy. However, MTX resistance (MTX-R) occurs in at least ≈33% of cases. This can sometimes be salvaged with actinomycin-D but often requires more toxic combination chemotherapy. Moreover, additional therapy may be needed and, for high-risk patients, 5% still die from the multidrug-resistant disease. Consequently, new treatments that are less toxic and could reverse MTX-R are needed. Here, we compared the proteome/phosphoproteome of MTX-resistant and sensitive choriocarcinoma cells using quantitative mass-spectrometry to identify therapeutically actionable molecular changes associated with MTX-R. Bioinformatics analysis of the proteomic data identified cell cycle and DNA damage repair as major pathways associated with MTX-R. MTX-R choriocarcinoma cells undergo cell cycle delay in G1 phase that enables them to repair DNA damage more efficiently through non-homologous end joining in an ATR-dependent manner. Increased expression of cyclin-dependent kinase 4 (CDK4) and loss of p16Ink4a in resistant cells suggested that CDK4 inhibition may be a strategy to treat MTX-R choriocarcinoma. Indeed, inhibition of CDK4/6 using genetic silencing or the clinically relevant inhibitor, Palbociclib, induced growth inhibition both in vitro and in an orthotopic in vivo mouse model. Finally, targeting the ATR pathway, genetically or pharmacologically, re-sensitised resistant cells to MTX in vitro and potently prevented the growth of MTX-R tumours in vivo. In short, we identified two novel therapeutic strategies to tackle MTX-R choriocarcinoma that could rapidly be translated into the clinic.
    DOI:  https://doi.org/10.1038/s41388-022-02251-8
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