bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒08‒30
forty-nine papers selected by
Sean Rudd
Karolinska Institutet


  1. Structure. 2020 Aug 15. pii: S0969-2126(20)30283-5. [Epub ahead of print]
    Eckenroth BE, Cao VB, Averill AM, Dragon JA, Doublié S.
      Oxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generating sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual "open" conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a "closed" conformation when bound to its substrate. Combined crystallographic and solution-scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of this enzyme. In addition, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.
    Keywords:  DNA damage; DNA repair; NEIL2 glycosylase; SAXS; X-ray crystallography
    DOI:  https://doi.org/10.1016/j.str.2020.08.001
  2. DNA Repair (Amst). 2020 Aug 14. pii: S1568-7864(20)30194-4. [Epub ahead of print]95 102945
    Melike Ç.
      DNA methylation on cytosine in CpG islands generates 5-methylcytosine (5mC), and further modification of 5mC can result in the oxidized variants 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxy (5caC). Base excision repair (BER) is crucial for both genome maintenance and active DNA demethylation of modified cytosine products and involves substrate-product channeling from nucleotide insertion by DNA polymerase (pol) β to the subsequent ligation step. Here, we report that, in contrast to the pol β mismatch insertion products (dCTP, dATP, and dTTP), the nicked products after pol β dGTP insertion can be ligated by DNA ligase I or DNA ligase III/XRCC1 complex when a 5mC oxidation modification is present opposite in the template position in vitro. A Pol β K280A mutation, which perturbates the stabilization of these base modifications within the active site, hinders the BER ligases. Moreover, the nicked repair intermediates that mimic pol β mismatch insertion products, i.e., with 3'-preinserted dGMP or dTMP opposite templating 5hmC, 5fC or 5caC, can be efficiently ligated, whereas preinserted 3'-dAMP or dCMP mismatches result in failed ligation reactions. These findings herein contribute to our understanding of the insertion tendencies of pol β opposite different cytosine base forms, the ligation properties of DNA ligase I and DNA ligase III/XRCC1 complex in the context of gapped and nicked damage-containing repair intermediates, and the efficiency and fidelity of substrate channeling during the final steps of BER in situations involving oxidative 5mC base modifications in the template strand.
    Keywords:  5-methylcytosine; Base excision repair; CpG; DNA ligase I; DNA ligase III; DNA methylation; DNA polymerase β; Islands
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102945
  3. EBioMedicine. 2020 Aug 23. pii: S2352-3964(20)30347-9. [Epub ahead of print]59 102971
    George SL, Lorenzi F, King D, Hartlieb S, Campbell J, Pemberton H, Toprak UH, Barker K, Tall J, da Costa BM, van den Boogaard ML, Dolman MEM, Molenaar JJ, Bryant HE, Westermann F, Lord CJ, Chesler L.
      BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research.METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF.
    FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft.
    INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic.
    FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.
    Keywords:  ATRX; DNA damage response; Neuroblastoma; PARP
    DOI:  https://doi.org/10.1016/j.ebiom.2020.102971
  4. Biochim Biophys Acta Gen Subj. 2020 Aug 25. pii: S0304-4165(20)30230-0. [Epub ahead of print] 129718
    Alekseeva IV, Kuznetsova AA, Bakman AS, Fedorova OS, Kuznetsov NA.
      BACKGROUND: Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5' side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA.METHODS: Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition.
    RESULTS: Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3'-5'-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre-steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied.
    CONCLUSIONS: It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme-substrate complex but did not affect the catalytic reaction.
    GENERAL SIGNIFICANCE: It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.
    Keywords:  Endoribonuclease activity; Fluorescence; Human apurinic/apyrimidinic endonuclease; Nucleotide recognition; Stopped-flow enzyme kinetics
    DOI:  https://doi.org/10.1016/j.bbagen.2020.129718
  5. Sci Adv. 2020 Jul;6(28): eaba5974
    Bennett LG, Wilkie AM, Antonopoulou E, Ceppi I, Sanchez A, Vernon EG, Gamble A, Myers KN, Collis SJ, Cejka P, Staples CJ.
      The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.
    DOI:  https://doi.org/10.1126/sciadv.aba5974
  6. Bioorg Med Chem. 2020 Sep 15. pii: S0968-0896(20)30491-0. [Epub ahead of print]28(18): 115661
    Arbour CA, Imperiali B.
      Nucleoside derivatives, in particular those featuring uridine, are familiar components of the nucleoside family of bioactive natural products. The structural complexity and biological activities of these compounds have inspired research from organic chemistry and chemical biology communities seeking to develop novel approaches to assemble the challenging molecular targets, to gain inspiration for enzyme inhibitor development and to fuel antibiotic discovery efforts. This review will present recent case studies describing the total synthesis and biosynthesis of uridine natural products, and de novo synthetic efforts exploiting features of the natural products to produce simplified scaffolds. This research has culminated in the development of complementary strategies that can lead to effective uridine-based inhibitors and antibiotics. The strengths and challenges of the juxtaposing methods will be illustrated by examining select uridine natural products. Moreover, structure-activity relationships (SAR) for each natural product-inspired scaffold will be discussed, highlighting the impact on inhibitor development, with the aim of future uridine-based small molecule expansion.
    Keywords:  Inhibitor discovery; Natural product antibiotics; Nucleoside analog; Structure-activity relationship; Uridine
    DOI:  https://doi.org/10.1016/j.bmc.2020.115661
  7. Genes (Basel). 2020 Aug 25. pii: E990. [Epub ahead of print]11(9):
    Zhu H, Swami U, Preet R, Zhang J.
      DNA replication is the fundamental process for accurate duplication and transfer of genetic information. Its fidelity is under constant stress from endogenous and exogenous factors which can cause perturbations that lead to DNA damage and defective replication. This can compromise genomic stability and integrity. Genomic instability is considered as one of the hallmarks of cancer. In normal cells, various checkpoints could either activate DNA repair or induce cell death/senescence. Cancer cells on the other hand potentiate DNA replicative stress, due to defective DNA damage repair mechanism and unchecked growth signaling. Though replicative stress can lead to mutagenesis and tumorigenesis, it can be harnessed paradoxically for cancer treatment. Herein, we review the mechanism and rationale to exploit replication stress for cancer therapy. We discuss both established and new approaches targeting DNA replication stress including chemotherapy, radiation, and small molecule inhibitors targeting pathways including ATR, Chk1, PARP, WEE1, MELK, NAE, TLK etc. Finally, we review combination treatments, biomarkers, and we suggest potential novel methods to target DNA replication stress to treat cancer.
    Keywords:  ATR; Chk1; DNA damage response; DNA replication stress; MELK; NAE; NEDD8; PARP; TLK; WEE1
    DOI:  https://doi.org/10.3390/genes11090990
  8. Nucleic Acids Res. 2020 Aug 28. pii: gkaa713. [Epub ahead of print]
    Korsholm LM, Gál Z, Nieto B, Quevedo O, Boukoura S, Lund CC, Larsen DH.
      DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.
    DOI:  https://doi.org/10.1093/nar/gkaa713
  9. Methods Mol Biol. 2021 ;2153 9-31
    Wong N, John S, Nussenzweig A, Canela A.
      DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.
    Keywords:  Adapter ligation; Agarose plugs; DNA damage; DNA double-strand breaks (DSBs); DNA repair; DSB mapping and quantification; End resection; Exonucleases; Next-generation sequencing; Nucleotide resolution
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_2
  10. Sci Adv. 2020 Jul;6(28): eaba7822
    Gao SS, Guan H, Yan S, Hu S, Song M, Guo ZP, Xie DF, Liu Y, Liu X, Zhang S, Zhou PK.
      Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.
    DOI:  https://doi.org/10.1126/sciadv.aba7822
  11. Gut. 2020 Aug 27. pii: gutjnl-2019-319984. [Epub ahead of print]
    Perkhofer L, Gout J, Roger E, Kude de Almeida F, Baptista Simões C, Wiesmüller L, Seufferlein T, Kleger A.
      Complex rearrangement patterns and mitotic errors are hallmarks of most pancreatic ductal adenocarcinomas (PDAC), a disease with dismal prognosis despite some therapeutic advances in recent years. DNA double-strand breaks (DSB) bear the greatest risk of provoking genomic instability, and DNA damage repair (DDR) pathways are crucial in preserving genomic integrity following a plethora of damage types. Two major repair pathways dominate DSB repair for safeguarding the genome integrity: non-homologous end joining and homologous recombination (HR). Defective HR, but also alterations in other DDR pathways, such as BRCA1, BRCA2, ATM and PALB2, occur frequently in both inherited and sporadic PDAC. Personalised treatment of pancreatic cancer is still in its infancy and predictive biomarkers are lacking. DDR deficiency might render a PDAC vulnerable to a potential new therapeutic intervention that increases the DNA damage load beyond a tolerable threshold, as for example, induced by poly (ADP-ribose) polymerase inhibitors. The Pancreas Cancer Olaparib Ongoing (POLO) trial, in which olaparib as a maintenance treatment improved progression-free survival compared with placebo after platinum-based induction chemotherapy in patients with PDAC and germline BRCA1/2 mutations, raised great hopes of a substantially improved outcome for this patient subgroup. This review summarises the relationship between DDR and PDAC, the prevalence and characteristics of DNA repair mutations and options for the clinical management of patients with PDAC and DNA repair deficiency.
    Keywords:  DNA damage; chemotherapy; pancreatic cancer
    DOI:  https://doi.org/10.1136/gutjnl-2019-319984
  12. Semin Cancer Biol. 2020 Aug 19. pii: S1044-579X(20)30177-2. [Epub ahead of print]
    Le Page C, Amuzu S, Rahimi K, Gotlieb W, Ragoussis J, Tonin PN.
      BRCA1 and BRCA2 are multi-functional proteins and key factors for maintaining genomic stability through their roles in DNA double strand break repair by homologous recombination, rescuing stalled or damaged DNA replication forks, and regulation of cell cycle DNA damage checkpoints. Impairment of any of these critical roles results in genomic instability, a phenotypic hallmark of many cancers including breast and epithelial ovarian carcinomas (EOC). Damaging, usually loss of function germline and somatic variants in BRCA1 and BRCA2, are important drivers of the development, progression, and management of high-grade serous tubo-ovarian carcinoma (HGSOC). However, mutations in these genes render patients particularly sensitive to platinum-based chemotherapy, and to the more innovative targeted therapies with poly-(ADP-ribose) polymerase inhibitors (PARPis) that are targeted to BRCA1/BRCA2 mutation carriers. Here, we reviewed the literature on the responsiveness of BRCA1/2-associated HGSOC to platinum-based chemotherapy and PARPis, and propose mechanisms underlying the frequent development of resistance to these therapeutic agents.
    Keywords:  BRCA; DNA repair; NHEJ; PARP inhibitors; Platinum; homologous recombination; ovarian cancer; replication fork
    DOI:  https://doi.org/10.1016/j.semcancer.2020.08.005
  13. Methods Mol Biol. 2021 ;2153 355-363
    Whelan DR, Rothenberg E.
      Single-molecule super-resolution microscopy (SRM) combines single-molecule detection with spatial resolutions tenfold improved over conventional confocal microscopy. These two key advantages make it possible to visualize individual DNA replication and damage events within the cellular context of fixed cells. This in turn engenders the ability to decipher variations between individual replicative and damage species within a single nucleus, elucidating different subpopulations of stress and repair events. Here, we describe the protocol for combining SRM with novel labeling and damage assays to characterize DNA double-strand break (DSB) induction at stressed replication forks (RFs) and subsequent repair by homologous recombination (HR). These assays enable spatiotemporal mapping of DNA damage response and repair proteins to establish their in vivo function and interactions, as well as detailed characterization of specific dysfunctions in HR caused by drugs or mutations of interest.
    Keywords:  DNA damage response; DNA double-strand break; DNA replication; Homologous recombination; Super resolution
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_24
  14. Sci Rep. 2020 Aug 28. 10(1): 14253
    Patidar PL, Viera T, Morales JC, Singh N, Motea EA, Khandelwal M, Fattah FJ.
      Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5'-3'-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.
    DOI:  https://doi.org/10.1038/s41598-020-71203-7
  15. Essays Biochem. 2020 Aug 26. pii: EBC20200016. [Epub ahead of print]
    MacDonald KM, Benguerfi S, Harding SM.
      Healthy cells experience thousands of DNA lesions per day during normal cellular metabolism, and ionizing radiation and chemotherapeutic drugs rely on DNA damage to kill cancer cells. In response to such lesions, the DNA damage response (DDR) activates cell-cycle checkpoints, initiates DNA repair mechanisms, or promotes the clearance of irreparable cells. Work over the past decade has revealed broader influences of the DDR, involving inflammatory gene expression following unresolved DNA damage, and immune surveillance of damaged or mutated cells. Subcellular structures called micronuclei, containing broken fragments of DNA or whole chromosomes that have been isolated away from the rest of the genome, are now recognized as one mediator of DDR-associated immune recognition. Micronuclei can initiate pro-inflammatory signaling cascades, or massively degrade to invoke distinct forms of genomic instability. In this mini-review, we aim to provide an overview of the current evidence linking the DDR to activation of the immune response through micronuclei formation, identifying key areas of interest, open questions, and emerging implications.
    Keywords:  Cell cycle checkpoints; DNA damage; Immune checkpoint blockade; Micronuclei; Radiotherapy
    DOI:  https://doi.org/10.1042/EBC20200016
  16. Methods Mol Biol. 2021 ;2153 307-328
    Osia B, Elango R, Kramara J, Roberts SA, Malkova A.
      Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.
    Keywords:  APOBEC; Break-induced replication; Contour-clamped homogenous electric field electrophoresis; Double-strand break; Gross chromosomal rearrangements; Homologous recombination; Single-stranded DNA
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_22
  17. Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31053-6. [Epub ahead of print]32(8): 108068
    Francica P, Mutlu M, Blomen VA, Oliveira C, Nowicka Z, Trenner A, Gerhards NM, Bouwman P, Stickel E, Hekkelman ML, Lingg L, Klebic I, van de Ven M, de Korte-Grimmerink R, Howald D, Jonkers J, Sartori AA, Fendler W, Chapman JR, Brummelkamp T, Rottenberg S.
      Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.
    DOI:  https://doi.org/10.1016/j.celrep.2020.108068
  18. DNA Repair (Amst). 2020 Aug 15. pii: S1568-7864(20)30195-6. [Epub ahead of print]95 102946
    Wang C, Chen Z, Su D, Tang M, Nie L, Zhang H, Feng X, Wang R, Shen X, Srivastava M, McLaughlin ME, Hart T, Li L, Chen J.
      Ataxia Telangiectasia and Rad3-Related kinase (ATR) is a master regulator of genome maintenance, and participates in DNA replication and various DNA repair pathways. In a genome-wide screen for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrates and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced interstrand crosslink (ICL) repair defect. We showed that C17orf53 is a ssDNA- and RPA-binding protein and both characteristics are important for its functions in the cell. In addition, using multiple omics methods, we found that C17orf53 works with MCM8/9 to promote cell survival in response to ICL lesions. Taken together, our data suggest that C17orf53 is a novel component involved in ICL repair pathway.
    Keywords:  C17orf53; DNA replication; Genome-wide screen; ICL repair; RPA-binding, HROB, MCM8IP; ssDNA-binding
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102946
  19. Methods Mol Biol. 2021 ;2153 535-554
    Piazza A, Rajput P, Heyer WD.
      DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.
    Keywords:  D-loop; DNA repair; Genomic instability; Homologous recombination; MIR; Multi-invasions
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_36
  20. Methods Mol Biol. 2021 ;2195 113-123
    Cavallo F, Caggiano C, Jasin M, Barchi M.
      Testicular germ cell tumors (TGCTs) are typically exquisitely sensitive to DNA interstrand cross-link (ICLs) agents. ICLs covalently link both strands of the DNA duplex, impeding fundamental cellular processes like DNA replication to cause cell death. A leading drug used for the treatment of TGCTs is cisplatin, which introduces ICLs and leads to formation of double strand breaks (DSBs), a DNA lesion that can be repaired in the S/G2 phases of the cell cycle by homologous recombination (HR, also termed homology-direct repair). Although most TGCTs respond to cisplatin-induced ICLs, a fraction is resistant to treatment. One proposed mechanism of TGCT resistance to cisplatin is an enhanced ability to repair DSBs by HR. Other than HR, repair of the ICL-lesions requires additional DNA repair mechanisms, whose action might also be implemented in therapy-resistant cells. This chapter describes GFP assays to measure (a) HR proficiency following formation of a DSB by the endonuclease I-SceI, and (b) HR repair induced by site-specific ICL formation involving psoralen. These experimental approaches can be used to determine the proficiency of TGCT cell lines in DSB repair by HR in comparison to HR repair of ICLs, providing tools to better characterize their recombination profile. Protocols of these assays have been adapted for use in Embryonal Carcinoma (EC) TGCT cell lines. Assays only require transient introduction of plasmids within cells, affording the advantage of testing multiple cell lines in a relatively short time.
    Keywords:  Embryonal carcinoma (EC); GFP reporters (DR-GFP, Tr-OriP-GFP); Homologous recombination; Interstrand cross-link (ICL) repair; Psoralen; TGCTs
    DOI:  https://doi.org/10.1007/978-1-0716-0860-9_9
  21. Methods Mol Biol. 2021 ;2153 59-69
    Zhou Y, Paull TT.
      DNA double-strand break (DSB) end resection initiates homologous recombination (HR) and is critical for genomic stability. DSB resection has been monitored indirectly in mammalian cells using detection of protein foci or BrdU foci formation, which is dependent on single-stranded DNA (ssDNA) products of resection. Here we describe a quantitative PCR (qPCR)-based assay to directly measure levels of ssDNA intermediates generated by resection at specific DSB sites in human cells, which is more quantitative and precise with respect to the extent and efficiency of resection compared with previous methods. This assay, excluding the time for making the stable cell line expressing the restriction enzyme AsiSI fused to the estrogen receptor hormone-binding domain (ER-AsiSI), can be completed within 3 days.
    Keywords:  DNA damage; DNA end resection; DNA repair; Single-stranded DNA quantitation
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_5
  22. Mol Cell. 2020 Aug 19. pii: S1097-2765(20)30548-7. [Epub ahead of print]
    Wang B, Grant RA, Laub MT.
      (p)ppGpp is a nucleotide messenger universally produced in bacteria following nutrient starvation. In E. coli, ppGpp inhibits purine nucleotide synthesis by targeting several different enzymes, but the physiological significance of their inhibition is unknown. Here, we report the structural basis of inhibition for one target, Gsk, the inosine-guanosine kinase. Gsk creates an unprecedented, allosteric binding pocket for ppGpp by restructuring terminal sequences, which restrains conformational dynamics necessary for catalysis. Guided by this structure, we generated a chromosomal mutation that abolishes Gsk regulation by ppGpp. This mutant strain accumulates abnormally high levels of purine nucleotides following amino-acid starvation, compromising cellular fitness. We demonstrate that this unrestricted increase in purine nucleotides is detrimental because it severely depletes pRpp and essential, pRpp-derived metabolites, including UTP, histidine, and tryptophan. Thus, our results reveal the significance of ppGpp's regulation of purine nucleotide synthesis and a critical mechanism by which E. coli coordinates biosynthetic processes during starvation.
    Keywords:  Gsk; amino-acid synthesis; pRpp; ppGpp; purine nucleotide synthesis; stringent response
    DOI:  https://doi.org/10.1016/j.molcel.2020.08.005
  23. Methods Mol Biol. 2021 ;2153 115-126
    Vugic D, Ehlén Å, Carreira A.
      DNA double-strand breaks (DSBs) are among the most toxic lesions. This type of DNA damage is repaired by two major pathways, homologous recombination (HR), operating only in S/G2 cell-cycle phases and nonhomologous end joining (NHEJ) which is operative throughout the cell cycle. Because HR is a template-directed repair, it is generally less prone to errors and/or translocations than NHEJ.The HR pathway involves several effector proteins and regulators that modulate the efficiency of repair and limit the repair outside S/G2 phase. Some of the genes coding for these proteins are frequently mutated in human diseases such as cancer, and pathogenic mutations or variants identified in patients often alter the HR proficiency of the cells.This chapter describes a cell-based gene-targeting reporter assay in human cells to evaluate the repair of a site-specific DSB by HR . In it, a promoter-less fluorescent protein is encoded in a plasmid flanked by two homology arms directed to a safe-harbour locus in the genome. The expression of the fluorescent protein is driven by the promoter of the endogenous locus enabling to quantify the efficiency of HR by flow cytometry. This approach can be used to determine the requirement of certain proteins, protein domains, or protein modifications for HR . It can also be used to functionally evaluate variants of the genes encoding these proteins such as BRCA1, BRCA2, RAD51C, and PALB2; which may help assess their pathogenicity. Here, we use the homologous recombination mediator BRCA2 to illustrate the assay.
    Keywords:  BRCA2; DNA double-strand breaks; Gene targeting; Homologous recombination; Template-directed DNA repair
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_9
  24. Methods Mol Biol. 2021 ;2153 427-438
    Arnould C, Rocher V, Legube G.
      Among the types of damage, DNA double-strand breaks (DSBs) (provoked by various environmental stresses, but also during normal cell metabolic activity) are the most deleterious, as illustrated by the variety of human diseases associated with DSB repair defects. DSBs are repaired by two groups of pathways: homologous recombination (HR) and nonhomologous end joining. These pathways do not trigger the same mutational signatures, and multiple factors, such as cell cycle stage, the complexity of the lesion and also the genomic location, contribute to the choice between these repair pathways. To study the usage of the HR machinery at DSBs, we propose a genome-wide method based on the chromatin immunoprecipitation of the HR core component Rad51, followed by high-throughput sequencing.
    Keywords:  ChIP-seq; Chromatin immunoprecipitation; DNA double-strand break; DNA repair; Homologous recombination; Next generation sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_29
  25. Methods Mol Biol. 2021 ;2153 447-458
    González-Prieto R, Cabello-Lobato MJ, Prado F.
      Homologous recombination (HR) has been extensively studied in response to DNA double-strand breaks (DSBs). In contrast, much less is known about how HR deals with DNA lesions other than DSBs (e.g., at single-stranded DNA) and replication forks, despite the fact that these DNA structures are associated with most spontaneous recombination events. A major handicap for studying the role of HR at non-DSB DNA lesions and replication forks is the difficulty of discriminating whether a recombination protein is associated with the non-DSB lesion per se or rather with a DSB generated during their processing. Here, we describe a method to follow the in vivo binding of recombination proteins to non-DSB DNA lesions and replication forks. This approach is based on the cleavage and subsequent electrophoretic analysis of the target DNA by the recombination protein fused to the micrococcal nuclease.
    Keywords:  2D DNA gel electrophoresis; Chromatin endogenous cleavage; DNA-binding protein; Non-double-strand break; Rad51; Rad52; Replication fork; Replication intermediates
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_31
  26. Methods Mol Biol. 2021 ;2153 383-393
    Tumini E, Aguilera A.
      The semiconservative nature of DNA replication allows the differential labeling of sister chromatids that is the fundamental requirement to perform the sister-chromatid exchange (SCE) assay. SCE assay is a powerful technique to visually detect the physical exchange of DNA between sister chromatids. SCEs could result as a consequence of DNA damage repair by homologous recombination (HR) during DNA replication. Here, we provide the detailed protocol to perform the SCE assay in cultured human cells. Cells are exposed to the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) during two cell cycles, resulting in the two sister chromatids having differential incorporation of the analog. After metaphase spreads preparation and further processing, SCEs are nicely visualized under the microscope.
    Keywords:  5-Bromo-2′-deoxyuridine (BrdU); DNA replication; Homologous recombination (HR); Human cells; Sister-chromatid exchange (SCE )
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_26
  27. Methods Mol Biol. 2021 ;2153 101-113
    Lahiri S, Jensen RB.
      The homologous recombination (HR) pathway maintains genomic integrity by repairing DNA double-strand breaks (DSBs), single-strand DNA gaps, and collapsed replication forks. The process of HR involves strand invasion, homology search, and DNA strand exchange between paired DNA molecules. HR is critical for the high-fidelity repair of DNA DSBs in mitotic cells and for the exchange of genetic information during meiosis. Here we describe a DNA strand exchange reaction in vitro utilizing purified proteins and defined DNA substrates to measure the strand invasion and pairing activities of the human RAD51 protein. We further discuss how this reaction can be catalytically stimulated by the mediator protein BRCA2.
    Keywords:  BRCA2; DNA double-strand breaks; DNA strand exchange; Genomic instability; Homologous recombination; RAD51; RPA; Strand invasion
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_8
  28. Methods Mol Biol. 2021 ;2153 365-381
    Kramarz K, Saada AA, Lambert SAE.
      The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.
    Keywords:  Benzoylated naphthoylated DEAE-cellulose; Bidimensional gel electrophoresis; Psoralen cross-links; Replication and recombination intermediates; Schizosaccharomyces pombe; Site-specific replication fork barrier
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_25
  29. Nat Commun. 2020 Aug 27. 11(1): 4287
    van Schie JJM, Faramarz A, Balk JA, Stewart GS, Cantelli E, Oostra AB, Rooimans MA, Parish JL, de Almeida Estéves C, Dumic K, Barisic I, Diderich KEM, van Slegtenhorst MA, Mahtab M, Pisani FM, Te Riele H, Ameziane N, Wolthuis RMF, de Lange J.
      Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.
    DOI:  https://doi.org/10.1038/s41467-020-18066-8
  30. Methods Mol Biol. 2021 ;2153 87-99
    Kwon Y, Sung P.
      Homologous recombination is a conserved genome maintenance pathway through which DNA double-strand breaks are eliminated and perturbed DNA replication forks and eroded telomeres are restored. The central step in homologous recombination is homology-dependent pairing between a single-stranded DNA tail with an intact duplex molecule to generate a displacement-loop (D-loop), followed by DNA synthesis within the D-loop platform. This chapter describes biochemical assays for (1) D-loop formation and DNA synthesis within the D-loop and (2) DNA strand displacement synthesis to test the role of DNA helicases (e.g., Pif1) in repair DNA synthesis. These mechanistic assays are valuable for elucidating the molecular details of HR.
    Keywords:  D-loop; DNA helicase; DNA polymerase; DNA synthesis; Homologous recombination; Recombinase
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_7
  31. Front Genet. 2020 ;11 855
    Velegzhaninov IO, Belykh ES, Rasova EE, Pylina YI, Shadrin DM, Klokov DY.
      Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.
    Keywords:  CRISPRa; DNA damage; DNA repair; RPA1 overexpression; radioresistance
    DOI:  https://doi.org/10.3389/fgene.2020.00855
  32. Br J Cancer. 2020 Aug 25.
    Le Tourneau C, Delord JP, Kotecki N, Borcoman E, Gomez-Roca C, Hescot S, Jungels C, Vincent-Salomon A, Cockenpot V, Eberst L, Molé A, Jdey W, Bono F, Trochon-Joseph V, Toussaint H, Zandanel C, Adamiec O, de Beaumont O, Cassier PA.
      BACKGROUND: AsiDNA, a first-in-class oligonucleotide-mimicking double-stranded DNA breaks, acts as a decoy agonist to DNA damage response in tumour cells. It also activates DNA-dependent protein kinase and poly (adenosine diphosphate [ADP]-ribose) polymerase enzymes that induce phosphorylation of H2AX and protein PARylation.METHODS: The aim of this Phase 1 study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), safety and pharmacokinetics/pharmacodynamics of AsiDNA administered daily for 3 days in the first week then weekly thereafter. Twenty-two patients with advanced solid tumours were enrolled in 5 dose levels: 200, 400, 600, 900, and 1300 mg, using a 3 + 3 design.
    RESULTS: The MTD was not reached. IV AsiDNA was safe. Two DLTs (grade 4 and grade 3 hepatic enzymes increased at 900 and 1300 mg), and two related SAE at 900 mg (grade 3 hypotension and grade 4 hepatic enzymes increased) were reported. AsiDNA PK increased proportionally with dose. A robust activation of DNA-PK by a significant posttreatment increase of γH2AX was evidenced in tumour biopsies.
    CONCLUSION: The dose of 600 mg was identified as the optimal dose for further clinical development.
    CLINICAL TRIAL REGISTRATION: Clinical trial registration (NCT number): NCT03579628.
    DOI:  https://doi.org/10.1038/s41416-020-01028-8
  33. Exp Hematol. 2020 Aug 25. pii: S0301-472X(20)30357-X. [Epub ahead of print]
    Khattar E, Tergaonkar V.
      Mammalian Rap1 is a part of the telomere binding complex named shelterin and it represents one of the most conserved telomeric protein. With its essential requirement in lower species to becoming necessary in higher species, it appears to have gained and lost several functions simultaneously evolving with telomeres. Mammalian Rap1 has been reported to play role in inflammation, metabolism as well as oxidative stress. Mammalian Rap1 has also been found to regulate DNA damage response from telomeres in senescent cells. Recently our group uncovered its novel role in stem cell maintenance, oncogenesis as well as its role in modulating chemotherapeutic response. Mechanistically it was shown to function as an adaptor via protein-protein interactions and modulate the response to DNA damage. In the current review we highlight newly identified functions of Rap1 in regulating telomeric and general DNA damage response with its impact at cellular and organismal level.
    Keywords:  DNA damage response; Rap1; cancer; chemotherapy; stem cells
    DOI:  https://doi.org/10.1016/j.exphem.2020.08.009
  34. Methods Mol Biol. 2021 ;2153 169-185
    Carreira R, Aguado FJ, Lama-Diaz T, Blanco MG.
      Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.
    Keywords:  DNA repair; Homologous recombination; Resolvase; Structure-selective endonucleases; Yen1/GEN1
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_12
  35. Methods Mol Biol. 2021 ;2153 329-353
    Willis NA, Scully R.
      Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.
    Keywords:  Flow cytometry; GFP; Homologous recombination; Long-tract gene conversion; Mouse embryonic stem cell; RFP; Replication fork barrier (RFB); Short-tract gene conversion; Sister-chromatid recombination; Tandem duplication; Tus-Ter
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_23
  36. Nat Commun. 2020 Aug 26. 11(1): 4263
    Schmidt JM, Bleichert F.
      Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.
    DOI:  https://doi.org/10.1038/s41467-020-18067-7
  37. Int J Biochem Cell Biol. 2020 Aug 23. pii: S1357-2725(20)30156-4. [Epub ahead of print] 105839
    Hristova RH, Stoynov SS, Tsaneva IR, Gospodinov AG.
      Chromatin regulators control transcription and replication, however if and how they might influence the coordination of these processes still is largely unknown. RUVBL1 and the related ATPase RUVBL2 participate in multiple nuclear processes and are implicated in cancer. Here, we report that both the excess and the deficit of the chromatin regulator RUVBL1 impede DNA replication as a consequence of altered transcription. Surprisingly, cells that either overexpressed or were silenced for RUVBL1 had slower replication fork rates and accumulated phosphorylated H2AX, dependent on active transcription. However, the mechanisms of transcription-dependent replication stress were different when RUVBL1 was overexpressed and when depleted. RUVBL1 overexpression led to increased c-Myc-dependent pause release of RNAPII, as evidenced by higher overall transcription, much stronger Ser2 phosphorylation of Rpb1- C-terminal domain, and enhanced colocalization of Rpb1 and c-Myc. RUVBL1 deficiency resulted in increased ubiquitination of Rpb1 and reduced mobility of an RNAP subunit, suggesting accumulation of stalled RNAPIIs on chromatin. Overall, our data show that by modulating the state of RNAPII complexes, RUVBL1 deregulation induces replication-transcription interference and compromises genome integrity during S-phase.
    Keywords:  DNA replication stress; DNA transcription; RUVBL1; c-Myc; replication-transcription conflicts
    DOI:  https://doi.org/10.1016/j.biocel.2020.105839
  38. Crit Rev Biochem Mol Biol. 2020 Aug 28. 1-26
    Caldwell CC, Spies M.
      The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.
    Keywords:  Conformational protein dynamics; DNA repair; DNA replication; homologous recombination; protein-DNA interctions; replication protein A (RPA); single-molecule
    DOI:  https://doi.org/10.1080/10409238.2020.1813070
  39. JCI Insight. 2020 Aug 25. pii: 140698. [Epub ahead of print]
    Golubicki M, Bonjoch L, Acuña-Ochoa JG, Díaz-Gay M, Muñoz J, Cuatrecasas M, Ocaña T, Iseas S, Mendez G, Cisterna D, Schubert SA, Nielsen M, van Wezel T, Goldberg Y, Pikarsky E, Robbio J, Roca E, Castells A, Balaguer F, Antelo M, Castellví-Bel S.
      Lynch syndrome is the most common colorectal cancer (CRC) hereditary form and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM, nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a LLS male patient with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR-Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than the MMR genes. We suggest MCM8 as a new gene involved in CRC germline predisposition with a recessive pattern of inheritance.
    Keywords:  Colorectal cancer; DNA repair; Gastroenterology; Genetic diseases; Genetics
    DOI:  https://doi.org/10.1172/jci.insight.140698
  40. DNA Repair (Amst). 2020 Aug 16. pii: S1568-7864(20)30196-8. [Epub ahead of print]95 102947
    Conti BA, Smogorzewska A.
      
    Keywords:  direct replication restart; fork reversal; re-priming; replication stress; template switching; translesion synthesis
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102947
  41. Methods Mol Biol. 2021 ;2153 1-8
    Barroso SI, Aguilera A.
      DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of genetic instability as they can lead to mutations, genome rearrangements, or loss of genetic material when not properly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBs with the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated in serine 139 in mammals (S129 in yeast), is named γ-H2AX. Detection of DSBs is of high relevance in research on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.
    Keywords:  DNA damage; Double-strand breaks; Immunoblotting; Immunofluorescence; γ-H2AX
    DOI:  https://doi.org/10.1007/978-1-0716-0644-5_1
  42. PLoS Genet. 2020 Aug 25. 16(8): e1008988
    Zhou Y, Pozo PN, Oh S, Stone HM, Cook JG.
      Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.
    DOI:  https://doi.org/10.1371/journal.pgen.1008988
  43. Biomed Res Int. 2020 ;2020 8916729
    Shan B, Zhao R, Zhou J, Zhang M, Qi X, Wang T, Gong J, Wu Y, Zhu Y, Yang W, Zhang Y, Wang G, Li X.
      AURKA, a cell cycle-regulated kinase, is associated with malignant transformation and progression in many cancer types. We analyzed the expression change of AURKA in pan-cancer and its effect on the prognosis of cancer patients using the TCGA dataset. We revealed that AURKA was extensively elevated and predicted a poor prognosis in most of the detected cancer types, with an exception in colon cancer. AURKA was elevated in colon cancer, but the upregulation of AURKA indicated better outcomes of colon cancer patients. Then we revealed that undermethylation of the AURKA gene and several transcription factors contributed to the upregulation of AURKA in colon cancer. Moreover, we demonstrated that AURKA overexpression promoted the death of colon cancer cells induced by Oxaliplatin, whereas knockdown of AURKA significantly weakened the chemosensitivity of colon cancer cells to Oxaliplatin. Mechanistically, AURKA inhibited DNA damage response by suppressing the expression of various DNA damage repair genes in a TP53-dependent manner, which can partly explain that ARUKA is associated with a beneficial outcome of colon cancer. This study provided a possibility to use AURKA as a biomarker to predict the chemosensitivity of colon cancer to platinum in the clinic.
    DOI:  https://doi.org/10.1155/2020/8916729
  44. Mol Genet Metab. 2020 Aug 05. pii: S1096-7192(20)30182-7. [Epub ahead of print]
    Shayota BJ, Donti TR, Xiao J, Gijavanekar C, Kennedy AD, Hubert L, Rodan L, Vanderpluym C, Nowak C, Bjornsson HT, Ganetzky R, Berry GT, Pappan KL, Sutton VR, Sun Q, Elsea SH.
      Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.
    Keywords:  Developmental delay; Inborn error of metabolism; Metabolome; Pentose phosphate pathway; Transaldolase deficiency; Transketolase deficiency
    DOI:  https://doi.org/10.1016/j.ymgme.2020.07.013
  45. Toxins (Basel). 2020 Aug 26. pii: E547. [Epub ahead of print]12(9):
    Mishima E, Ichijo M, Kawabe T, Kikuchi K, Akiyama Y, Toyohara T, Suzuki T, Suzuki C, Asao A, Ishii N, Fukuda S, Abe T.
      Alterations in microbiota are known to affect kidney disease conditions. We have previously shown that germ-free conditions exacerbated adenine-induced kidney damage in mice; however, the mechanism by which this occurs has not been elucidated. To explore this mechanism, we examined the influence of germ-free conditions on purine metabolism and renal immune responses involved in the kidney damage. Germ-free mice showed higher expression levels of purine-metabolizing enzymes such as xanthine dehydrogenase, which converts adenine to a nephrotoxic byproduct 2,8-dihydroxyadenine (2,8-DHA). The germ-free mice also showed increased urinary excretion of allantoin, indicating enhanced purine metabolism. Metabolome analysis demonstrated marked differences in the purine metabolite levels in the feces of germ-free mice and mice with microbiota. Furthermore, unlike the germ-free condition, antibiotic treatment did not increase the expression of purine-metabolizing enzymes or exacerbate adenine-induced kidney damage. Considering renal immune responses, the germ-free mice displayed an absence of renal IL-17A expression. However, the adenine-induced kidney damage in wild-type mice was comparable to that in IL-17A-deficient mice, suggesting that IL-17A does not play a major role in the disease condition. Our results suggest that the enhanced host purine metabolism in the germ-free mice potentially promotes the conversion of the administered adenine into 2,8-DHA, resulting in exacerbated kidney damage. This further suggests a role of the microbiota in regulating host purine metabolism.
    Keywords:  IL-17; Th17; chronic kidney disease; gut-kidney axis; microbiota; uremic toxins; uric acids; xanthine dehydrogenase; xanthine oxidase
    DOI:  https://doi.org/10.3390/toxins12090547
  46. Sci Rep. 2020 Aug 24. 10(1): 14092
    Chen Y, Ni J, Gao Y, Zhang J, Liu X, Chen Y, Chen Z, Wu Y.
      Colorectal cancer (CRC) is a common malignancy occurring in the digestive system. Despite progress in surgery and therapy options, CRC is still a considerable cause of cancer mortality worldwide. In this study, a colon cancer patient-derived xenograft model was established to evaluate the antitumor activity of Shikonin. The protective effect underlying Shikonin was determined through assessing serum levels of liver enzymes (ALT, AST) and kidney functions (BuN, Scr) in PDX mice. Proteomics and metabolomics profiles were integrated to provide a systematic perspective in dynamic changes of proteins and global endogenous metabolites as well as their perturbed pathways. A total of 456 differently expressed proteins (DEPs), 32 differently expressed metabolites (DEMs) in tumor tissue, and 20 DEMs in mice serum were identified. The perturbation of arginine biosynthesis, purine metabolism, and biosynthesis of amino acids may mainly account for therapeutic mechanism of Shikonin. Furthermore, the expression of mRNAs participating in arginine biosynthesis (CPS1, OTC, Arg1) and do novo purine synthesis (GART, PAICS, ATIC) were validated through RT-qPCR. Our study provides new insights into the drug therapeutic strategies and a better understanding of antitumor mechanisms that might be valuable for further studies on Shikonin in the clinical treatment of colorectal cancer.
    DOI:  https://doi.org/10.1038/s41598-020-71116-5
  47. Analyst. 2020 Aug 27.
    Hu C, Jiang K, Shao Z, Shi M, Meng HM.
      Guanosine-5'-triphosphate (GTP) plays a key role in many important biological processes of cells. It is not only a primer for DNA replication and one of the four essential nucleoside triphosphates for mRNA synthesis, but also an energy source for translation and other important cellular processes. It can be converted to adenine nucleoside triphosphate (ATP), and the intracellular GTP level is closely related to the specific pathological state, so it is crucial to establish a simple and accurate method for the detection of GTP. Deoxyribozymes have unique catalytic and structural properties. One of the deoxyribozymes which is named DK2 with self-phosphorylation ability can transfer a phosphate from GTP to the 5' end in the presence of manganese(ii), while lambda exonuclease (λexo) catalyzes the gradual hydrolysis of double-stranded DNA molecules phosphorylated at the 5'-end from 5' to 3', but cannot cleave the 5'-OH end. The fluorescent dye SYBR Green I (SG I) can bind to dsDNA and produce significant fluorescence, but it can only give out weak fluorescence when it is mixed with a single strand. Here, we present a novel unlabeled fluorescence assay for GTP based on the self-phosphorylation of deoxyribozyme DK2 and the specific hydrolysis of λexo. Owing to the advantages of simple operation, high sensitivity, good specificity, low cost and without fluorophore (quenching group) labeling, this method has great potential in biological applications.
    DOI:  https://doi.org/10.1039/d0an01334j
  48. Front Cell Infect Microbiol. 2020 ;10 391
    Hyeon S, Lee MK, Kim YE, Lee GM, Ahn JH.
      Sterile alpha motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) acts as a restriction factor for several RNA and DNA viruses by limiting the intracellular pool of deoxynucleoside triphosphates. Here, we investigated the regulation of SAMHD1 expression during human cytomegalovirus (HCMV) infection. SAMHD1 knockdown using shRNA increased the activity of the viral UL99 late gene promoter in human fibroblasts by 7- to 9-fold, confirming its anti-HCMV activity. We also found that the level of SAMHD1 was initially increased by HCMV infection but decreased partly at the protein level at late stages of infection. SAMHD1 loss was not observed with UV-inactivated virus and required viral DNA replication. This reduction of SAMHD1 was effectively blocked by MLN4924, an inhibitor of the Cullin-RING-E3 ligase (CRL) complexes, but not by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPase. Indirect immunofluorescence assays further supported the CRL-mediated SAMHD1 loss at late stages of virus infection. Knockdown of CUL2 and to a lesser extent CUL1 using siRNA stabilized SAMHD1 in normal fibroblasts and inhibited SAMHD1 loss during virus infection. Altogether, our results demonstrate that SAMHD1 inhibits the growth of HCMV, but HCMV causes degradation of SAMHD1 at late stages of viral infection through the CRL complexes.
    Keywords:  CRL; HCMV; SAMHD1; degradation; restriction factor
    DOI:  https://doi.org/10.3389/fcimb.2020.00391
  49. Arab J Gastroenterol. 2020 Aug 20. pii: S1687-1979(20)30077-0. [Epub ahead of print]
    Ramadan RA, Moghazy TF, Hafez R, Morsi H, Samir M, Shamesya M.
      BACKGROUND AND STUDY AIMS: The reprogramming of metabolic pathways in tumour cells is a crucial step to meet the increased requirements for their own growth. This process occurs through alterations in gene expression, polymorphisms, and epigenetic dysregulation of a number of metabolic genes. Several metabolic enzymatic pathways such as pyrimidine-metabolizing enzymes have been implicated in tumorigenesis and tumor progression.PATIENTS AND METHODS: We measured the relative expression levels of three pyrimidine-metabolizing genes-thymidylate synthase (TYMS), thymidine phosphorylase (TYMP), and dihydropyrimidine dehydrogenase (DPYD)-in tumor tissue and adjacent normal-appearing mucosa in 50 colon cancer (CC) patients using real-time reverse-transcription polymerase chain reaction. Gene expression was also studied in relation to demographic and pathological criteria.
    RESULTS: The gene expression levels of both TYMS and TYMP were significantly higher in tumor tissue than normal adjacent tissue. Further, they showed an agreeable level of diagnostic performance as a means to discriminate between normal and tumor tissue; TYMS had high specificity (94%) but moderate sensitivity (60%), while TYPM showed average sensitivity (70%) and specificity (76%). Although DPYD expression was lower in tumor tissue than paracancerous tissue, this level did not reach the statistical significance. TYMS expression was significantly higher in moderately and poorly differentiated tumors than in well-differentiated ones. There was no significant association between gene expression and the remaining clinicopathological criteria (e.g., age, sex, tumor location, and metastasis). We found a positive correlation between the gene expression levels of TYMS and DPYD.
    CONCLUSION: TYMS and TYMP messenger RNA levels seem to be plausible indicators in the diagnosis of CC, although further studies are warranted for validation.
    Keywords:  Colon cancer; Dihydropyrimidine dehydrogenase; Gene expression; Real-time reverse-transcription polymerase chain reaction; Thymidine phosphorylase; Thymidylate synthase
    DOI:  https://doi.org/10.1016/j.ajg.2020.07.006