bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021–10–03
forty-four papers selected by
Sean Rudd, Karolinska Institutet
  1. The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair.
  2. Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair.
  3. A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination.
  4. DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.
  5. Protein-Protein Interactions in Translesion Synthesis.
  6. The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks.
  7. DNA Damage Response in Glioblastoma: Mechanism for Treatment Resistance and Emerging Therapeutic Strategies.
  8. Determinants of Homologous Recombination Deficiency in Pancreatic Cancer.
  9. A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.
  10. Determination of DNA lesion bypass using a ChIP-based assay.
  11. Integration of DNA damage responses with dynamic spatial genome organization.
  12. MTH1 inhibitor TH1579 induces oxidative DNA damage and mitotic arrest in acute myeloid leukemia.
  13. Tackling PARP inhibitor resistance.
  14. Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae.
  15. Towards a CRISPeR understanding of homologous recombination with high-throughput functional genomics.
  16. Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11.
  17. p53 mRNA Metabolism Links with the DNA Damage Response.
  18. Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast.
  19. DNArepairK: An Interactive Database for Exploring the Impact of Anticancer Drugs onto the Dynamics of DNA Repair Proteins.
  20. The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.
  21. DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection.
  22. The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response.
  23. Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair.
  24. Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance.
  25. PARP Inhibitors in Melanoma-An Expanding Therapeutic Option?
  26. The Role of the Rad55-Rad57 Complex in DNA Repair.
  27. Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models.
  28. Deposition Bias of Chromatin Proteins Inverts under DNA Replication Stress Conditions.
  29. Recognition and removal of clustered DNA lesions via nucleotide excision repair.
  30. Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome.
  31. ATM's Role in the Repair of DNA Double-Strand Breaks.
  32. The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks.
  33. New Methodologies to Study DNA Repair Processes in Space and Time Within Living Cells.
  34. SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.
  35. TP53 loss initiates chromosomal instability in fallopian tube epithelial cells.
  36. Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.
  37. One-step enzymatic labeling reveals a critical role of O-GlcNAcylation in cell-cycle progression and DNA damage response.
  38. Replication stress inhibits synthesis of histone mRNAs in yeast by removing Spt10p and Spt21p from the histone promoters.
  39. miR-27b-3p a Negative Regulator of DSB-DNA Repair.
  40. Supercoiling and looping promote DNA base accessibility and coordination among distant sites.
  41. BRCA1 prevents R-loop-associated centromeric instability.
  42. Histone deacetylase 6 acts upstream of DNA damage response activation to support the survival of glioblastoma cells.
  43. Recent Updates on Mechanisms of Resistance to 5-Fluorouracil and Reversal Strategies in Colon Cancer Treatment.
  44. Structural basis for isoform-specific inhibition of human CTPS1.
  1. Mol Cell. 2021 Sep 28. pii: S1097-2765(21)00742-5. [Epub ahead of print]
    The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair.
    Richard O Adeyemi, Nicholas A Willis, Andrew E H Elia, Connor Clairmont, Shibo Li, Xiaohua Wu, Alan D D'Andrea, Ralph Scully, Stephen J Elledge.
      Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.
    Keywords:  CRISPR; EXO1; FANCM; Protexin; REV3L; SCAI; homologous recombination; inter-strand crosslinks; replication stress; resection
    DOI:  https://doi.org/10.1016/j.molcel.2021.09.008
  2. Genes (Basel). 2021 Aug 25. pii: 1310. [Epub ahead of print]12(9):
    Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair.
    Karl E Zahn, Ryan B Jensen.
      The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5'-dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5'-resected substrates by locating and pairing microhomologies present in 3'-overhanging single-stranded tails, cleaving the extraneous 3'-DNA by dNTP-dependent end-processing, before extending the nascent 3' end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions.
    Keywords:  53BP1 effector; 5′-end resection; DNA repair; PARP-i; POLQ; cancer; polymerase θ; polθ-i; shieldin; synthetic lethality
    DOI:  https://doi.org/10.3390/genes12091310
  3. Nat Commun. 2021 Sep 30. 12(1): 5748
    A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination.
    Sara M Ambjørn, Julien P Duxin, Emil P T Hertz, Isha Nasa, Joana Duro, Thomas Kruse, Blanca Lopez-Mendez, Beata Rymarczyk, Lauren E Cressey, Thomas van Overeem Hansen, Arminja N Kettenbach, Vibe H Oestergaard, Michael Lisby, Jakob Nilsson.
      Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
    DOI:  https://doi.org/10.1038/s41467-021-26079-0
  4. EMBO J. 2021 Sep 29. e103787
    DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.
    Laurent Cappadocia, Tomasz Kochańczyk, Christopher D Lima.
      Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.
    Keywords:  E3 ligase; homologous recombination; post-translational modification; replication protein A; small ubiquitin-like modifier
    DOI:  https://doi.org/10.15252/embj.2019103787
  5. Molecules. 2021 Sep 13. pii: 5544. [Epub ahead of print]26(18):
    Protein-Protein Interactions in Translesion Synthesis.
    Radha Charan Dash, Kyle Hadden.
      Translesion synthesis (TLS) is an error-prone DNA damage tolerance mechanism used by actively replicating cells to copy past DNA lesions and extend the primer strand. TLS ensures that cells continue replication in the presence of damaged DNA bases, albeit at the expense of an increased mutation rate. Recent studies have demonstrated a clear role for TLS in rescuing cancer cells treated with first-line genotoxic agents by allowing them to replicate and survive in the presence of chemotherapy-induced DNA lesions. The importance of TLS in both the initial response to chemotherapy and the long-term development of acquired resistance has allowed it to emerge as an interesting target for small molecule drug discovery. Proper TLS function is a complicated process involving a heteroprotein complex that mediates multiple attachment and switching steps through several protein-protein interactions (PPIs). In this review, we briefly describe the importance of TLS in cancer and provide an in-depth analysis of key TLS PPIs, focusing on key structural features at the PPI interface while also exploring the potential druggability of each key PPI.
    Keywords:  REV1; cancer; polymerase ζ; protein–protein interaction; translesion synthesis
    DOI:  https://doi.org/10.3390/molecules26185544
  6. Genes (Basel). 2021 Sep 16. pii: 1430. [Epub ahead of print]12(9):
    The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks.
    Ian Yannuzzi, Margaret A Butler, Joel Fernandez, Jeannine R LaRocque.
      DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.
    Keywords:  CtIP; Drosophila; double-strand break repair; end resection; homologous recombination; non-homologous end-joining; single-strand annealing
    DOI:  https://doi.org/10.3390/genes12091430
  7. Cancer J. 2021 Sep-Oct 01;27(5):27(5): 379-385
    DNA Damage Response in Glioblastoma: Mechanism for Treatment Resistance and Emerging Therapeutic Strategies.
    Alipi Bonm, Santosh Kesari.
       ABSTRACT: Glioblastoma (GBM) is an intrinsically treatment-resistant tumor and has been shown to upregulate DNA damage response (DDR) components after treatment. DNA damage response signaling mediates treatment resistance by promoting cell cycle arrest in order to allow for DNA damage repair and avoid mitotic catastrophe. Therefore, targeting the DDR pathway is an attractive strategy to combat treatment resistance in GBM. In this review, we discuss the different DDR pathways and then summarize the current preclinical evidence for DDR inhibitors in GBM, as well as completed and ongoing clinical trials.
    DOI:  https://doi.org/10.1097/PPO.0000000000000540
  8. Cancers (Basel). 2021 Sep 21. pii: 4716. [Epub ahead of print]13(18):
    Determinants of Homologous Recombination Deficiency in Pancreatic Cancer.
    Max M Wattenberg, Kim A Reiss.
      Pancreatic cancer is a treatment-resistant malignancy associated with high mortality. However, defective homologous recombination (HR), a DNA repair mechanism required for high-fidelity repair of double-strand DNA breaks, is a therapeutic vulnerability. Consistent with this, a subset of patients with pancreatic cancer show unique tumor responsiveness to HR-dependent DNA damage triggered by certain treatments (platinum chemotherapy and PARP inhibitors). While pathogenic mutations in HR genes are a major driver of this sensitivity, another layer of diverse tumor intrinsic and extrinsic factors regulate the HR deficiency (HRD) phenotype. Defining the mechanisms that drive HRD may guide the development of novel strategies and therapeutics to induce treatment sensitivity in non-HRD tumors. Here, we discuss the complexity underlying HRD in pancreatic cancer and highlight implications for identifying and treating this distinct subset of patients.
    Keywords:  DNA damage repair; homologous recombination deficiency; pancreatic cancer
    DOI:  https://doi.org/10.3390/cancers13184716
  9. EMBO J. 2021 Sep 27. e108439
    A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.
    Verena Hurst, Kiran Challa, Felix Jonas, Romain Forey, Ragna Sack, Jan Seebacher, Christoph D Schmid, Naama Barkai, Kenji Shimada, Susan M Gasser, Jérôme Poli.
      Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.
    Keywords:  Mec1; nuclear pore; replication checkpoint; replication interference; replication stress; transcription
    DOI:  https://doi.org/10.15252/embj.2021108439
  10. DNA Repair (Amst). 2021 Sep 22. pii: S1568-7864(21)00186-5. [Epub ahead of print]108 103230
    Determination of DNA lesion bypass using a ChIP-based assay.
    Dayong Wu, Ananya Banerjee, Shurui Cai, Na Li, Chunhua Han, Xuetao Bai, Junran Zhang, Qi-En Wang.
      DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.
    Keywords:  Cancer stem cell; ChIP; Chromatin immunoprecipitation; Cisplatin; Lesion bypass; TLS; Translesion synthesis
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103230
  11. Trends Genet. 2021 Sep 28. pii: S0168-9525(21)00261-4. [Epub ahead of print]
    Integration of DNA damage responses with dynamic spatial genome organization.
    Mia Stanic, Karim Mekhail.
      The maintenance of genome stability and cellular homeostasis depends on the temporal and spatial coordination of successive events constituting the classical DNA damage response (DDR). Recent findings suggest close integration and coordination of DDR signaling with specific cellular processes. The mechanisms underlying such coordination remain unclear. We review emerging crosstalk between DNA repair factors, chromatin remodeling, replication, transcription, spatial genome organization, cytoskeletal forces, and liquid-liquid phase separation (LLPS) in mediating DNA repair. We present an overarching DNA repair framework within which these dynamic processes intersect in nuclear space over time. Collectively, this interplay ensures the efficient assembly of DNA repair proteins onto shifting genome structures to preserve genome stability and cell survival.
    Keywords:  DNA damage response; genome organization; nucleus; spatiotemporal; transcription
    DOI:  https://doi.org/10.1016/j.tig.2021.08.016
  12. Cancer Res. 2021 Sep 30. pii: canres.0061.2021. [Epub ahead of print]
    MTH1 inhibitor TH1579 induces oxidative DNA damage and mitotic arrest in acute myeloid leukemia.
    Kumar Sanjiv, José Manuel Calderón-Montaño, Therese M Pham, Tom Erkers, Viktoriia Tsuber, Ingrid Almlöf, Andreas Höglund, Yaser Heshmati, Brinton Seashore-Ludlow, Akhilesh Nagesh Danda, Helge Gad, Elisee Wiita, Camilla Göktürk, Azita Rasti, Stefanie Friedrich, Anders Centio, Montserrat Estruch, Thea Kristin Våtsveen, Nona Struyf, Torkild Visnes, Martin Scobie, Tobias Koolmeister, Martin Henriksson, Olov Wallner, Teresa Sandvall, Sören Lehmann, Kim Theilgaard-Mönch, Mathew J Garnett, Päivi Östling, Julian Walfridsson, Thomas Helleday, Ulrika Warpman Berglund.
      Acute myeloid leukemia (AML) is an aggressive hematological malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematological cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-),which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the pre-clinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rational to investigate the clinical usefulness of TH1579 in AML in an on-going clinical phase 1 trial.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0061
  13. Trends Cancer. 2021 Sep 22. pii: S2405-8033(21)00175-8. [Epub ahead of print]
    Tackling PARP inhibitor resistance.
    Kasper Fugger, Graeme Hewitt, Stephen C West, Simon J Boulton.
      Homologous recombination-deficient (HRD) tumours, including those harbouring mutations in the BRCA genes, are hypersensitive to treatment with inhibitors of poly(ADP-ribose) polymerase (PARPis). Despite high response rates, most HRD cancers ultimately develop resistance to PARPi treatment through reversion mutations or genetic/epigenetic alterations to DNA repair pathways. Counteracting these resistance pathways, thereby increasing the potency of PARPi therapy, represents a potential strategy to improve the treatment of HRD cancers. In this review, we discuss recent insights derived from genetic screens that have identified a number of novel genes that can be targeted to improve PARPi treatment of HRD cancers and may provide a means to overcome PARPi resistance.
    Keywords:  base excision repair; homologous recombination repair; nucleotide metabolism; poly(ADP-ribose) polymerase inhibitors; therapy resistance
    DOI:  https://doi.org/10.1016/j.trecan.2021.08.007
  14. Cells. 2021 Sep 03. pii: 2309. [Epub ahead of print]10(9):
    Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae.
    Stanislav G Kozmin, Gregory Eot-Houllier, Anne Reynaud-Angelin, Didier Gasparutto, Evelyne Sage.
      Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.
    Keywords:  base excision repair; clustered DNA damage; ionizing radiation; multiply damaged sites; mutagenesis; mutagenic potential; non-DSB clustered DNA damage; oxidized base lesions
    DOI:  https://doi.org/10.3390/cells10092309
  15. Curr Opin Genet Dev. 2021 Sep 25. pii: S0959-437X(21)00106-4. [Epub ahead of print]71 171-181
    Towards a CRISPeR understanding of homologous recombination with high-throughput functional genomics.
    Samuel B Hayward, Alberto Ciccia.
      CRISPR-dependent genome editing enables the study of genes and mutations on a large scale. Here we review CRISPR-based functional genomics technologies that generate gene knockouts and single nucleotide variants (SNVs) and discuss how their use has provided new important insights into the function of homologous recombination (HR) genes. In particular, we highlight discoveries from CRISPR screens that have contributed to define the response to PARP inhibition in cells deficient for the HR genes BRCA1 and BRCA2, uncover genes whose loss causes synthetic lethality in combination with BRCA1/2 deficiency, and characterize the function of BRCA1/2 SNVs of uncertain clinical significance. Further use of these approaches, combined with next-generation CRISPR-based technologies, will aid to dissect the genetic network of the HR pathway, define the impact of HR mutations on cancer etiology and treatment, and develop novel targeted therapies for HR-deficient tumors.
    DOI:  https://doi.org/10.1016/j.gde.2021.08.006
  16. Cancers (Basel). 2021 Sep 14. pii: 4601. [Epub ahead of print]13(18):
    Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11.
    Ukhyun Jo, Yasuhisa Murai, Naoko Takebe, Anish Thomas, Yves Pommier.
      Precision medicine aims to implement strategies based on the molecular features of tumors and optimized drug delivery to improve cancer diagnosis and treatment. DNA replication is a logical approach because it can be targeted by a broad range of anticancer drugs that are both clinically approved and in development. These drugs increase deleterious replication stress (RepStress); however, how to selectively target and identify the tumors with specific molecular characteristics are unmet clinical needs. Here, we provide background information on the molecular processes of DNA replication and its checkpoints, and discuss how to target replication, checkpoint, and repair pathways with ATR inhibitors and exploit Schlafen 11 (SLFN11) as a predictive biomarker.
    Keywords:  ATR; Adavoserib; Camptothecin; Ceralasertib; PARP; Rucaparib; berzosertib; cisplatin; niraparib; olaparib; replication stress; schlafen 11; talazoparib; temozolomide
    DOI:  https://doi.org/10.3390/cancers13184601
  17. Genes (Basel). 2021 Sep 20. pii: 1446. [Epub ahead of print]12(9):
    p53 mRNA Metabolism Links with the DNA Damage Response.
    Sivakumar Vadivel Gnanasundram, Ondrej Bonczek, Lixiao Wang, Sa Chen, Robin Fahraeus.
      Human cells are subjected to continuous challenges by different genotoxic stress attacks. DNA damage leads to erroneous mutations, which can alter the function of oncogenes or tumor suppressors, resulting in cancer development. To circumvent this, cells activate the DNA damage response (DDR), which mainly involves cell cycle regulation and DNA repair processes. The tumor suppressor p53 plays a pivotal role in the DDR by halting the cell cycle and facilitating the DNA repair processes. Various pathways and factors participating in the detection and repair of DNA have been described, including scores of RNA-binding proteins (RBPs) and RNAs. It has become increasingly clear that p53's role is multitasking, and p53 mRNA regulation plays a prominent part in the DDR. This review is aimed at covering the p53 RNA metabolism linked to the DDR and highlights the recent findings.
    Keywords:  ATM kinase; DNA damage response; MDM2; RNA metabolism; RNA-binding proteins; mRNA translation; p53
    DOI:  https://doi.org/10.3390/genes12091446
  18. G3 (Bethesda). 2021 Sep 27. pii: jkab245. [Epub ahead of print]11(10):
    Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast.
    Joseph A Stewart, Michael B Hillegass, Joseph H Oberlitner, Ellen M Younkin, Beth F Wasserman, Anne M Casper.
      Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field electrophoresis, next-generation whole-genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or "late" template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.
    Keywords:  BIR; LOH; LTGC; fragile site; gap repair; gene conversion; half crossover; homologous recombination; loss of heterozygosity; replication fork collapse; replication stress; template switching
    DOI:  https://doi.org/10.1093/g3journal/jkab245
  19. Biomedicines. 2021 Sep 16. pii: 1238. [Epub ahead of print]9(9):
    DNArepairK: An Interactive Database for Exploring the Impact of Anticancer Drugs onto the Dynamics of DNA Repair Proteins.
    Yordan Babukov, Radoslav Aleksandrov, Aneliya Ivanova, Aleksandar Atemin, Stoyno Stoynov.
      Cells are constantly exposed to numerous mutagens that produce diverse types of DNA lesions. Eukaryotic cells have evolved an impressive array of DNA repair mechanisms that are able to detect and repair these lesions, thus preventing genomic instability. The DNA repair process is subjected to precise spatiotemporal coordination, and repair proteins are recruited to lesions in an orderly fashion, depending on their function. Here, we present DNArepairK, a unique open-access database that contains the kinetics of recruitment and removal of 70 fluorescently tagged DNA repair proteins to complex DNA damage sites in living HeLa Kyoto cells. An interactive graphical representation of the data complemented with live cell imaging movies facilitates straightforward comparisons between the dynamics of proteins contributing to different DNA repair pathways. Notably, most of the proteins included in DNArepairK are represented by their kinetics in both nontreated and PARP1/2 inhibitor-treated (talazoparib) cells, thereby providing an unprecedented overview of the effects of anticancer drugs on the regular dynamics of the DNA damage response. We believe that the exclusive dataset available in DNArepairK will be of value to scientists exploring the DNA damage response but, also, to inform and guide the development and evaluation of novel DNA repair-targeting anticancer drugs.
    Keywords:  CRC mathematical modeling; DNA damage response; DNA repair protein kinetics; DNArepairK database; PARP1/2 inhibitors; anticancer drug development; laser micro-irradiation; live cell imaging; talazoparib
    DOI:  https://doi.org/10.3390/biomedicines9091238
  20. Cell Rep. 2021 Sep 28. pii: S2211-1247(21)01210-9. [Epub ahead of print]36(13): 109756
    The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.
    Wanyu Bai, Guangchao Zhu, Jiejie Xu, Pingyue Chen, Feilong Meng, Hongman Xue, Chun Chen, Junchao Dong.
      Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3' single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3' flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3' single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4-/- cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.
    Keywords:  3′ flap endonuclease; 53BP1; DNA ligase 4; DSB repair; XPF-ERCC1; alternative end joining; chromosomal translocation; class switch recombination
    DOI:  https://doi.org/10.1016/j.celrep.2021.109756
  21. Mol Cell Biol. 2021 Sep 27. MCB0005621
    DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection.
    Salar Ahmad, Valérie Côté, Jacques Côté.
      The repair of DNA double-strand breaks (DSBs) occurs in chromatin and several histone post-translational modifications have been implicated in the process. Modifications of histone H2A N-terminal tail has also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γH2A) and mutant mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1ATM and Mec1ATR, and loss of Lcd1ATRIP or Mec1 signaling decreases γH2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This is supported by an increase of RPA and a decrease in DNA signal near the DSB in the H2AS-15E phosphomimic mutant, indicating higher resection. This serine is replaced by a lysine in mammals (H2AK15), which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different post-translational modifications between organisms but achieving the same function.
    DOI:  https://doi.org/10.1128/MCB.00056-21
  22. Int J Mol Sci. 2021 Sep 14. pii: 9900. [Epub ahead of print]22(18):
    The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response.
    Siti A M Imran, Muhammad Dain Yazid, Wei Cui, Yogeswaran Lokanathan.
      Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.
    Keywords:  DNA damage response; TRF2; extra-telomeric; telomeres protection
    DOI:  https://doi.org/10.3390/ijms22189900
  23. Biomolecules. 2021 Aug 27. pii: 1284. [Epub ahead of print]11(9):
    Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair.
    Anzhela V Pavlova, Elena A Kubareva, Mayya V Monakhova, Maria I Zvereva, Nina G Dolinnaya.
      DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.
    Keywords:  DNA damage; DNA repair; G-quadruplex; G-quadruplex-binding proteins; genomic instability
    DOI:  https://doi.org/10.3390/biom11091284
  24. Cancers (Basel). 2021 Sep 07. pii: 4501. [Epub ahead of print]13(18):
    Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance.
    Marcin Herok, Bartosz Wawrzynow, Marta J Maluszek, Maciej B Olszewski, Alicja Zylicz, Maciej Zylicz.
      Analyzing the TCGA breast cancer database, we discovered that patients with the HER2 cancer subtype and overexpression of MDM2 exhibited decreased post-treatment survival. Inhibition of MDM2 expression in the SKBR3 cell line (HER2 subtype) diminished the survival of cancer cells treated with doxorubicin, etoposide, and camptothecin. Moreover, we demonstrated that inhibition of MDM2 expression diminished DNA repair by homologous recombination (HR) and sensitized SKBR3 cells to a PARP inhibitor, olaparib. In H1299 (TP53-/-) cells treated with neocarzinostatin (NCS), overexpression of MDM2 WT or E3-dead MDM2 C478S variant stimulated the NCS-dependent phosphorylation of ATM, NBN, and BRCA1, proteins involved in HR DNA repair. However, overexpression of chaperone-dead MDM2 K454A variant diminished phosphorylation of these proteins as well as the HR DNA repair. Moreover, we demonstrated that, upon NCS treatment, MDM2 K454A interacted with NBN more efficiently than MDM2 WT and that MDM2 WT was degraded more efficiently than MDM2 K454A. Using a proliferation assay, we showed that overexpression of MDM2 WT, but not MDM2 K454A, led to acquisition of resistance to NCS. The presented results indicate that, following chemotherapy, MDM2 WT was released from MDM2-NBN complex and efficiently degraded, hence allowing extensive HR DNA repair leading to the acquisition of chemoresistance by cancer cells.
    Keywords:  DNA damage repair; MDM2; NBN; chemoresistance; homologous recombination
    DOI:  https://doi.org/10.3390/cancers13184501
  25. Cancers (Basel). 2021 Sep 08. pii: 4520. [Epub ahead of print]13(18):
    PARP Inhibitors in Melanoma-An Expanding Therapeutic Option?
    Wei Yen Chan, Lauren J Brown, Lee Reid, Anthony M Joshua.
      Immunotherapy has transformed the treatment landscape of melanoma; however, despite improvements in patient outcomes, monotherapy can often lead to resistance and tumour escape. Therefore, there is a need for new therapies, combination strategies and biomarker-guided decision making to increase the subset of patients most likely to benefit from treatment. Poly (ADP-ribose) polymerase (PARP) inhibitors act by synthetic lethality to target tumour cells with homologous recombination deficiencies such as BRCA mutations. However, the application of PARP inhibitors could be extended to a broad range of BRCA-negative cancers with high rates of DNA damage repair pathway mutations, such as melanoma. Additionally, PARP inhibition has the potential to augment the therapeutic effect of immunotherapy through multi-faceted immune-priming capabilities. In this review, we detail the immunological role of PARP and rationale for combining PARP and immune checkpoint inhibitors, with a particular focus on a subset of melanoma with homologous recombination defects that may benefit most from this targeted approach. We summarise the biology supporting this combined regimen and discuss preclinical results as well as ongoing clinical trials in melanoma which may impact future treatment.
    Keywords:  DNA damage response; PARP inhibitor; combination therapy; homologous recombination; immunotherapy; melanoma
    DOI:  https://doi.org/10.3390/cancers13184520
  26. Genes (Basel). 2021 Sep 08. pii: 1390. [Epub ahead of print]12(9):
    The Role of the Rad55-Rad57 Complex in DNA Repair.
    Upasana Roy, Eric C Greene.
      Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55-Rad57 in HR.
    Keywords:  Rad51; Rad51 paralogs; Rad55–Rad57; homologous recombination; recombination mediators
    DOI:  https://doi.org/10.3390/genes12091390
  27. Int J Mol Sci. 2021 Sep 09. pii: 9766. [Epub ahead of print]22(18):
    Hydroxygenkwanin Increases the Sensitivity of Liver Cancer Cells to Chemotherapy by Inhibiting DNA Damage Response in Mouse Xenograft Models.
    Chin-Chuan Chen, Chi-Yuan Chen, Shu-Fang Cheng, Tzong-Ming Shieh, Yann-Lii Leu, Wen-Yu Chuang, Kuang-Ting Liu, Shir-Hwa Ueng, Yin-Hwa Shih, Li-Fang Chou, Tong-Hong Wang.
      Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.
    Keywords:  DNA damage response; RAD51; homologous recombination; hydroxygenkwanin; liver cancer
    DOI:  https://doi.org/10.3390/ijms22189766
  28. ACS Chem Biol. 2021 Sep 30.
    Deposition Bias of Chromatin Proteins Inverts under DNA Replication Stress Conditions.
    Martijn R H Zwinderman, Thamar Jessurun Lobo, Petra E van der Wouden, Diana C J Spierings, Marcel A T M van Vugt, Peter M Lansdorp, Victor Guryev, Frank J Dekker.
      Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.
    DOI:  https://doi.org/10.1021/acschembio.1c00321
  29. DNA Repair (Amst). 2021 Sep 10. pii: S1568-7864(21)00181-6. [Epub ahead of print]108 103225
    Recognition and removal of clustered DNA lesions via nucleotide excision repair.
    N V Naumenko, I O Petruseva, A A Lomzov, O I Lavrik.
      Clustered damage of DNA consists of two or more lesions located within one or two turns of the DNA helix. Clusters consisting of lesions of various structures can arise under the influence of strong damaging factors, especially if the cells have a compromised repair status. In this work, we analyzed how the presence of an analog of the apurinic/apyrimidinic site - a non-nucleoside residue consisting of diethylene glycol phosphodiester (DEG) - affects the recognition and removal of a bulky lesion (a non-nucleoside site of the modified DNA strand containing a fluorescein residue, nFlu) from DNA by a mammalian nucleotide excision repair system. Here we demonstrated that the efficiency of nFlu removal decreases in the presence of DEG in the complementary strand and is completely suppressed when the DEG is located opposite the nFlu. By contrast, protein factor XPC-RAD23B, which initiates global genomic nucleotide excision repair, has higher affinity for DNA containing clustered damage as compared to DNA containing a single bulky lesion; the affinity of XPC strengthens as the positions of DEG and nFlu become closer. The changes in the double-stranded DNA's geometry caused by the presence of clustered damage were also assessed. The obtained experimental data together with the results of molecular dynamics simulations make it possible to get insight into the structural features of DNA containing clustered lesions that determine the efficiency of repair. Speaking more broadly, this study should help to understand the probable fate of bulky adduct-containing clusters of various topologies in the mammalian cell.
    Keywords:  Clustered DNA lesions; Higher eukaryote; Molecular dynamics; Multiple DNA lesions; Nucleotide excision repair
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103225
  30. Front Genet. 2021 ;12 708860
    Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome.
    Rachel Adihe Lokanga, Daman Kumari, Karen Usdin.
      The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.
    Keywords:  MUS81/EME1; R-loops; SLX1-SLX4; break-induced DNA replication; mitotic DNA synthesis; origins of replication; replication fork blockage; secondary DNA structures
    DOI:  https://doi.org/10.3389/fgene.2021.708860
  31. Genes (Basel). 2021 Aug 31. pii: 1370. [Epub ahead of print]12(9):
    ATM's Role in the Repair of DNA Double-Strand Breaks.
    Atsushi Shibata, Penny A Jeggo.
      Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage-e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair.
    Keywords:  ATM; DNA double-strand break; homologous recombination; ionizing radiation; non-homologous end joining
    DOI:  https://doi.org/10.3390/genes12091370
  32. Genes (Basel). 2021 Sep 15. pii: 1415. [Epub ahead of print]12(9):
    The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks.
    Apfrida Kendek, Marieke R Wensveen, Aniek Janssen.
      The eukaryotic nucleus is continuously being exposed to endogenous and exogenous sources that cause DNA breaks, whose faithful repair requires the activity of dedicated nuclear machineries. DNA is packaged into a variety of chromatin domains, each characterized by specific molecular properties that regulate gene expression and help maintain nuclear structure. These different chromatin environments each demand a tailored response to DNA damage. Silenced chromatin domains in particular present a major challenge to the cell's DNA repair machinery due to their specific biophysical properties and distinct, often repetitive, DNA content. To this end, we here discuss the interplay between silenced chromatin domains and DNA damage repair, specifically double-strand breaks, and how these processes help maintain genome stability.
    Keywords:  DNA-damage repair; constitutive heterochromatin; double-strand breaks; facultative heterochromatin
    DOI:  https://doi.org/10.3390/genes12091415
  33. Front Cell Dev Biol. 2021 ;9 730998
    New Methodologies to Study DNA Repair Processes in Space and Time Within Living Cells.
    Siham Zentout, Rebecca Smith, Marine Jacquier, Sébastien Huet.
      DNA repair requires a coordinated effort from an array of factors that play different roles in the DNA damage response from recognizing and signaling the presence of a break, creating a repair competent environment, and physically repairing the lesion. Due to the rapid nature of many of these events, live-cell microscopy has become an invaluable method to study this process. In this review we outline commonly used tools to induce DNA damage under the microscope and discuss spatio-temporal analysis tools that can bring added information regarding protein dynamics at sites of damage. In particular, we show how to go beyond the classical analysis of protein recruitment curves to be able to assess the dynamic association of the repair factors with the DNA lesions as well as the target-search strategies used to efficiently find these lesions. Finally, we discuss how the use of mathematical models, combined with experimental evidence, can be used to better interpret the complex dynamics of repair proteins at DNA lesions.
    Keywords:  DNA damage; fluorescence fluctuation analysis; kinetic modeling; live cell imaging; spatio-temporal analysis
    DOI:  https://doi.org/10.3389/fcell.2021.730998
  34. Nat Commun. 2021 Oct 01. 12(1): 5779
    SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.
    Jian Ma, Qing Shi, Gaofeng Cui, Haoyue Sheng, Maria Victoria Botuyan, Yingke Zhou, Yuqian Yan, Yundong He, Liguo Wang, Yuzhuo Wang, Georges Mer, Dingwei Ye, Chenji Wang, Haojie Huang.
      Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.
    DOI:  https://doi.org/10.1038/s41467-021-26049-6
  35. Dis Model Mech. 2021 Sep 27. pii: dmm.049001. [Epub ahead of print]
    TP53 loss initiates chromosomal instability in fallopian tube epithelial cells.
    Daniel Bronder, Anthony Tighe, Darawalee Wangsa, Dali Zong, Thomas J Meyer, René Wardenaar, Paul Minshall, Daniela Hirsch, Kerstin Heselmeyer-Haddad, Louisa Nelson, Diana Spierings, Joanne C McGrail, Maggie Cam, André Nussenzweig, Floris Foijer, Thomas Ried, Stephen S Taylor.
      High-grade serous ovarian cancer (HGSOC) originates in the fallopian tube epithelium and is characterized by ubiquitous TP53 mutation and extensive chromosomal instability (CIN). However, direct causes of CIN, such as mutations in DNA replication and mitosis genes, are rare in HGSOC. We therefore asked whether oncogenic mutations that are common in HGSOC can indirectly drive CIN in non-transformed human fallopian tube epithelial cells. To model homologous recombination deficient HGSOC, we sequentially mutated TP53 and BRCA1 then overexpressed MYC. Loss of p53 function alone was sufficient to drive the emergence of sub-clonal karyotype alterations. TP53 mutation also led to global gene expression changes, influencing modules involved in cell cycle commitment, DNA replication, G2/M checkpoint control, and mitotic spindle function. Both transcriptional deregulation and karyotype diversity were exacerbated by loss of BRCA1 function, with whole-genome doubling events observed in independent p53/BRCA1-deficient lineages. Thus, our observations indicate that loss of the key tumour suppressor TP53 is sufficient to deregulate multiple cell cycle control networks and thereby initiate CIN in pre-malignant fallopian tube epithelial cells.
    Keywords:  BRCA1; Chromosomal instability; Fallopian tube; High-grade serous ovarian cancer; MYC; TP53
    DOI:  https://doi.org/10.1242/dmm.049001
  36. Mol Biol Rep. 2021 Oct 02.
    Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.
    Ajanta Chatterji, Arne Holmgren, Rajib Sengupta.
      Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.
    Keywords:  Glutaredoxin; Glutathione; Ribonucleotide reductases; S-glutathionylation
    DOI:  https://doi.org/10.1007/s11033-021-06721-2
  37. Angew Chem Int Ed Engl. 2021 Sep 30.
    One-step enzymatic labeling reveals a critical role of O-GlcNAcylation in cell-cycle progression and DNA damage response.
    Yinping Tian, Qiang Zhu, Zeyu Sun, Didi Geng, Bingyi Lin, Xiaoling Su, Jiahui He, Miao Guo, Hong Xu, Ye Zhao, Weijie Qin, Peng George Wang, Liuqing Wen, Wen Yi.
      O-linked N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous post-translational modification of proteins that is essential for cell function. Perturbation of O-GlcNAcylation leads to altered cell-cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one-step enzymatic strategy for capture and profiling O-GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O-GlcNAcylation. FEN1 O-GlcNAcylation is dynamically regulated during the cell cycle. O-GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O-GlcNAcylated proteins, and reveals an unknown mechanism of O-GlcNAcylation in regulating cell cycle progression and DNA damage response.
    Keywords:  DNA damage response; FEN1; O-GlcNAcylation; cell cycle; chemoenzymatic labelling
    DOI:  https://doi.org/10.1002/anie.202110053
  38. J Biol Chem. 2021 Sep 25. pii: S0021-9258(21)01049-8. [Epub ahead of print] 101246
    Replication stress inhibits synthesis of histone mRNAs in yeast by removing Spt10p and Spt21p from the histone promoters.
    Madhura Bhagwat, Shreya Nagar, Pritpal Kaur, Riddhi Mehta, Ivana Vancurova, Ales Vancura.
      Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometry for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In mammalian cells, replication stress triggers degradation of histone mRNAs, but it is unclear if this mechanism is conserved from other species. The aim of this study was to identify the histone mRNA decay pathway in the yeast Saccharomyces cerevisiae and determine the mechanism by which DNA replication stress represses histone mRNAs. Using RT-qPCR and ChIP-qPCR, we show here that histone mRNAs can be degraded by both 5' → 3' and 3' → 5' pathways; however, replication stress does not trigger decay of histone mRNA in yeast. Rather, replication stress inhibits transcription of histone genes by removing the histone genes-specific transcription factors Spt10p and Spt21p from histone promoters, leading to disassembly of the preinitiation complexes and eviction of RNA Pol II from histone genes by a mechanism facilitated by checkpoint kinase Rad53p and histone chaperone Asf1p. In contrast, replication stress does not remove SBF transcription complex, another activator of histone genes, from the histone promoters, suggesting that Spt10p and Spt21p have unique roles in the transcriptional downregulation of histone genes during replication stress. Together, our data show that, unlike in mammalian cells, replication stress in yeast does not trigger decay of histone mRNAs but inhibits histone transcription.
    Keywords:  Replication stress; chromatin; histone mRNA; mRNA decay; promoter; transcription
    DOI:  https://doi.org/10.1016/j.jbc.2021.101246
  39. Genes (Basel). 2021 Aug 27. pii: 1333. [Epub ahead of print]12(9):
    miR-27b-3p a Negative Regulator of DSB-DNA Repair.
    Ricardo I Peraza-Vega, Mahara Valverde, Emilio Rojas.
      Understanding the regulation of DNA repair mechanisms is of utmost importance to identify altered cellular processes that lead to diseases such as cancer through genomic instability. In this sense, miRNAs have shown a crucial role. Specifically, miR-27b-3 biogenesis has been shown to be induced in response to DNA damage, suggesting that this microRNA has a role in DNA repair. In this work, we show that the overexpression of miR-27b-3p reduces the ability of cells to repair DNA lesions, mainly double-stranded breaks (DSB), and causes the deregulation of genes involved in homologous recombination repair (HRR), base excision repair (BER), and the cell cycle. DNA damage was induced in BALB/c-3T3 cells, which overexpress miR-27b-3p, using xenobiotic agents with specific mechanisms of action that challenge different repair mechanisms to determine their reparative capacity. In addition, we evaluated the expression of 84 DNA damage signaling and repair genes and performed pathway enrichment analysis to identify altered cellular processes. Taken together, our results indicate that miR-27b-3p acts as a negative regulator of DNA repair when overexpressed.
    Keywords:  DNA repair; cancer; comet assay; double-strand break; gene regulation; miR-27b-3p
    DOI:  https://doi.org/10.3390/genes12091333
  40. Nat Commun. 2021 Sep 28. 12(1): 5683
    Supercoiling and looping promote DNA base accessibility and coordination among distant sites.
    Jonathan M Fogg, Allison K Judge, Erik Stricker, Hilda L Chan, Lynn Zechiedrich.
      DNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity.
    DOI:  https://doi.org/10.1038/s41467-021-25936-2
  41. Cell Death Dis. 2021 Oct 01. 12(10): 896
    BRCA1 prevents R-loop-associated centromeric instability.
    Carine Racca, Sébastien Britton, Sabrine Hédouin, Claire Francastel, Patrick Calsou, Florence Larminat.
      Centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.
    DOI:  https://doi.org/10.1038/s41419-021-04189-3
  42. Cell Death Dis. 2021 Sep 28. 12(10): 884
    Histone deacetylase 6 acts upstream of DNA damage response activation to support the survival of glioblastoma cells.
    Wen-Bin Yang, An-Chih Wu, Tsung-I Hsu, Jing-Ping Liou, Wei-Lun Lo, Kwang-Yu Chang, Pin-Yuan Chen, Ushio Kikkawa, Shung-Tai Yang, Tzu-Jen Kao, Ruei-Ming Chen, Wen-Chang Chang, Chiung-Yuan Ko, Jian-Ying Chuang.
      DNA repair promotes the progression and recurrence of glioblastoma (GBM). However, there remain no effective therapies for targeting the DNA damage response and repair (DDR) pathway in the clinical setting. Thus, we aimed to conduct a comprehensive analysis of DDR genes in GBM specimens to understand the molecular mechanisms underlying treatment resistance. Herein, transcriptomic analysis of 177 well-defined DDR genes was performed with normal and GBM specimens (n = 137) from The Cancer Genome Atlas and further integrated with the expression profiling of histone deacetylase 6 (HDAC6) inhibition in temozolomide (TMZ)-resistant GBM cells and patient-derived tumor cells. The effects of HDAC6 inhibition on DDR signaling were examined both in vitro and intracranial mouse models. We found that the expression of DDR genes, involved in repair pathways for DNA double-strand breaks, was upregulated in highly malignant primary and recurrent brain tumors, and their expression was related to abnormal clinical features. However, a potent HDAC6 inhibitor, MPT0B291, attenuated the expression of these genes, including RAD51 and CHEK1, and was more effective in blocking homologous recombination repair in GBM cells. Interestingly, it resulted in lower cytotoxicity in primary glial cells than other HDAC6 inhibitors. MPT0B291 reduced the growth of both TMZ-sensitive and TMZ-resistant tumor cells and prolonged survival in mouse models of GBM. We verified that HDAC6 regulated DDR genes by affecting Sp1 expression, which abolished MPT0B291-induced DNA damage. Our findings uncover a regulatory network among HDAC6, Sp1, and DDR genes for drug resistance and survival of GBM cells. Furthermore, MPT0B291 may serve as a potential lead compound for GBM therapy.
    DOI:  https://doi.org/10.1038/s41419-021-04182-w
  43. Biology (Basel). 2021 Aug 31. pii: 854. [Epub ahead of print]10(9):
    Recent Updates on Mechanisms of Resistance to 5-Fluorouracil and Reversal Strategies in Colon Cancer Treatment.
    Shamin Azwar, Heng Fong Seow, Maha Abdullah, Mohd Faisal Jabar, Norhafizah Mohtarrudin.
      5-Fluorouracil (5-FU) plus leucovorin (LV) remain as the mainstay standard adjuvant chemotherapy treatment for early stage colon cancer, and the preferred first-line option for metastatic colon cancer patients in combination with oxaliplatin in FOLFOX, or irinotecan in FOLFIRI regimens. Despite treatment success to a certain extent, the incidence of chemotherapy failure attributed to chemotherapy resistance is still reported in many patients. This resistance, which can be defined by tumor tolerance against chemotherapy, either intrinsic or acquired, is primarily driven by the dysregulation of various components in distinct pathways. In recent years, it has been established that the incidence of 5-FU resistance, akin to multidrug resistance, can be attributed to the alterations in drug transport, evasion of apoptosis, changes in the cell cycle and DNA-damage repair machinery, regulation of autophagy, epithelial-to-mesenchymal transition, cancer stem cell involvement, tumor microenvironment interactions, miRNA dysregulations, epigenetic alterations, as well as redox imbalances. Certain resistance mechanisms that are 5-FU-specific have also been ascertained to include the upregulation of thymidylate synthase, dihydropyrimidine dehydrogenase, methylenetetrahydrofolate reductase, and the downregulation of thymidine phosphorylase. Indeed, the successful modulation of these mechanisms have been the game plan of numerous studies that had employed small molecule inhibitors, plant-based small molecules, and non-coding RNA regulators to effectively reverse 5-FU resistance in colon cancer cells. It is hoped that these studies would provide fundamental knowledge to further our understanding prior developing novel drugs in the near future that would synergistically work with 5-FU to potentiate its antitumor effects and improve the patient's overall survival.
    Keywords:  5-FU; 5-fluorouracil; chemotherapy drug resistance; colon cancer; dihydropyrimidine dehydrogenase; methylenetetrahydrofolate reductase; overcoming chemotherapy drug resistance; thymidine phosphorylase; thymidylate synthase
    DOI:  https://doi.org/10.3390/biology10090854
  44. Proc Natl Acad Sci U S A. 2021 Oct 05. pii: e2107968118. [Epub ahead of print]118(40):
    Structural basis for isoform-specific inhibition of human CTPS1.
    Eric M Lynch, Michael A DiMattia, Steven Albanese, Gydo C P van Zundert, Jesse M Hansen, Joel D Quispe, Madison A Kennedy, Andreas Verras, Kenneth Borrelli, Angela V Toms, Neelu Kaila, Kevin D Kreutter, Joshua J McElwee, Justin M Kollman.
      Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
    Keywords:  CTP synthase; cryo-EM; enzymes; immune disorders; small molecules
    DOI:  https://doi.org/10.1073/pnas.2107968118
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