bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒01‒17
thirty-one papers selected by
Sean Rudd
Karolinska Institutet

  1. Cancer Cell. 2021 Jan 13. pii: S1535-6108(20)30658-9. [Epub ahead of print]
    Kalev P, Hyer ML, Gross S, Konteatis Z, Chen CC, Fletcher M, Lein M, Aguado-Fraile E, Frank V, Barnett A, Mandley E, Goldford J, Chen Y, Sellers K, Hayes S, Lizotte K, Quang P, Tuncay Y, Clasquin M, Peters R, Weier J, Simone E, Murtie J, Liu W, Nagaraja R, Dang L, Sui Z, Biller SA, Travins J, Marks KM, Marjon K.
      The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
    Keywords:  DNA damage; Fanconi anemia complex; MAT2A; PRMT5; R loops; detained introns; splicing; synergy; taxanes
  2. Mol Cell. 2021 Jan 11. pii: S1097-2765(20)30946-1. [Epub ahead of print]
    Nakamura K, Kustatscher G, Alabert C, Hödl M, Forne I, Völker-Albert M, Satpathy S, Beyer TE, Mailand N, Choudhary C, Imhof A, Rappsilber J, Groth A.
      Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
    Keywords:  ATM; BRCA1-A; Camptothecin; NDRG3; NHEJ; PLK1; UBAP2; homologous recombination; nascent chromatin capture; replication stress
  3. Mol Cell. 2021 Jan 04. pii: S1097-2765(20)30939-4. [Epub ahead of print]
    Belan O, Barroso C, Kaczmarczyk A, Anand R, Federico S, O'Reilly N, Newton MD, Maeots E, Enchev RI, Martinez-Perez E, Rueda DS, Boulton SJ.
      Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
    Keywords:  BRCA2; DNA repair; Rad51 nucleoprotein filaments; Rad51 paralogs; homologous recombination; single molecule approaches
  4. Redox Biol. 2021 Jan 02. pii: S2213-2317(20)31053-3. [Epub ahead of print]40 101848
    Zhang L, Misiara L, Samaranayake GJ, Sharma N, Nguyen DM, Tahara YK, Kool ET, Rai P.
      Cancer cells develop protective adaptations against oxidative DNA damage, providing a strong rationale for targeting DNA repair proteins. There has been a high degree of recent interest in inhibiting the mammalian Nudix pyrophosphatase MutT Homolog 1 (MTH1). MTH1 degrades 8-oxo-dGTP, thus limiting its incorporation into genomic DNA. MTH1 inhibition has variously been shown to induce genomic 8-oxo-dG elevation, genotoxic strand breaks in p53-functional cells, and tumor-inhibitory outcomes. Genomically incorporated 8-oxo-dG is excised by the base excision repair enzyme, 8-oxo-dG glycosylase 1 (OGG1). Thus, OGG1 inhibitors have been developed with the idea that their combination with MTH1 inhibitors will have anti-tumor effects by increasing genomic oxidative DNA damage. However, contradictory to this idea, we found that human lung adenocarcinoma with low OGG1 and MTH1 were robustly represented in patient datasets. Furthermore, OGG1 co-depletion mitigated the extent of DNA strand breaks and cellular senescence in MTH1-depleted p53-wildtype lung adenocarcinoma cells. Similarly, shMTH1-transduced cells were less sensitive to the OGG1 inhibitor, SU0268, than shGFP-transduced counterparts. Although the dual OGG1/MTH1 inhibitor, SU0383, induced greater cytotoxicity than equivalent combined or single doses of its parent scaffold MTH1 and OGG1 inhibitors, IACS-4759 and SU0268, this effect was only observed at the highest concentration assessed. Collectively, using both genetic depletion as well as small molecule inhibitors, our findings suggest that OGG1/MTH1 co-inhibition is unlikely to yield significant tumor-suppressive benefit. Instead such co-inhibition may exert tumor-protective effects by preventing base excision repair-induced DNA nicks and p53 induction, thus potentially conferring a survival advantage to the treated tumors.
    Keywords:  MTH1 inhibition; OGG1 inhibition; lung cancer; oxidative DNA damage; p53; senescence
  5. FEBS Lett. 2021 Jan 10.
    Nolan M, Knudson K, Holz MK, Chaudhury I.
      We have previously demonstrated that Fanconi Anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.
  6. Proc Natl Acad Sci U S A. 2021 Jan 19. pii: e2010370118. [Epub ahead of print]118(3):
    Elbakry A, Juhász S, Chan KC, Löbrich M.
      Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.
    Keywords:  DNA repair synthesis; Holliday junctions; crossovers; sister chromatid exchanges; synthesis-dependent strand annealing
  7. Genes Dev. 2021 Jan 14.
    Li S, Bonner JN, Wan B, So S, Summers A, Gonzalez L, Xue X, Zhao X.
      SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.
    Keywords:  Esc2; SUMO E2; genome maintenance; homologous recombination
  8. Cell Rep. 2021 Jan 12. pii: S2211-1247(20)31614-4. [Epub ahead of print]34(2): 108625
    Wang J, Oh YT, Li Z, Dou J, Tang S, Wang X, Wang H, Takeda S, Wang Y.
      Radiation sensitive 52 (RAD52) is an important factor for double-strand break repair (DSBR). However, deficiency in vertebrate/mammalian Rad52 has no apparent phenotype. The underlying mechanism remains elusive. Here, we report that RAD52 deficiency increased cell survival after camptothecin (CPT) treatment. CPT generates single-strand breaks (SSBs) that further convert to double-strand breaks (DSBs) if they are not repaired. RAD52 inhibits SSB repair (SSBR) through strong single-strand DNA (ssDNA) and/or poly(ADP-ribose) (PAR) binding affinity to reduce DNA-damage-promoted X-Ray Repair Cross Complementing 1 (XRCC1)/ligase IIIα (LIG3α) co-localization. The inhibitory effects of RAD52 on SSBR neutralize the role of RAD52 in DSBR, suggesting that RAD52 may maintain a balance between cell survival and genomic integrity. Furthermore, we demonstrate that blocking RAD52 oligomerization that disrupts RAD52's DSBR, while retaining its ssDNA binding capacity that is required for RAD52's inhibitory effects on SSBR, sensitizes cells to different DNA-damaging agents. This discovery provides guidance for developing efficient RAD52 inhibitors in cancer therapy.
    Keywords:  DNA damage; DNA repair; RAD52; XRCC1; single-strand breaks
  9. Front Genet. 2020 ;11 607428
    Yue X, Bai C, Xie D, Ma T, Zhou PK.
      DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a member of the phosphatidylinositol 3-kinase related kinase family, which can phosphorylate more than 700 substrates. As the core enzyme, DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to play crucial roles in cellular DNA damage response (DDR). Once DNA double strand breaks (DSBs) occur in the cells, DNA-PKcs is promptly recruited into damage sites and activated. DNA-PKcs is auto-phosphorylated and phosphorylated by Ataxia-Telangiectasia Mutated at multiple sites, and phosphorylates other targets, participating in a series of DDR and repair processes, which determine the cells' fates: DSBs NHEJ repair and pathway choice, replication stress response, cell cycle checkpoints, telomeres length maintenance, senescence, autophagy, etc. Due to the special and multi-faceted roles of DNA-PKcs in the cellular responses to DNA damage, it is important to precisely regulate the formation and dynamic of its functional complex and activities for guarding genomic stability. On the other hand, targeting DNA-PKcs has been considered as a promising strategy of exploring novel radiosensitizers and killing agents of cancer cells. Combining DNA-PKcs inhibitors with radiotherapy can effectively enhance the efficacy of radiotherapy, offering more possibilities for cancer therapy.
    Keywords:  DNA damage response; DNA repair; DNA-PKcs; genomic instability; radiosensitization
  10. J Mol Biol. 2021 Jan 12. pii: S0022-2836(20)30731-2. [Epub ahead of print] 166806
    Ramdzan ZM, Vickridge E, Li L, Faraco CCF, Djerir B, Leduy L, Maréchal A, Nepveu A.
      The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.
    Keywords:  Base excision repair; CUT domains; CUX1; DNA polymerase β; abasic sites; cisplatin resistance; deoxyribose (dRP)-lyase; intrastrand G-crosslinks
  11. Nat Protoc. 2021 Jan 13.
    Kit Leng Lui S, Keegan S, Tonzi P, Kahli M, Chen YH, Chalhoub N, Coleman KE, Fenyo D, Smith DJ, Huang TT.
      The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.
  12. Nucleic Acids Res. 2021 Jan 15. pii: gkaa1265. [Epub ahead of print]
    Mehnert AK, Prorocic M, Dujeancourt-Henry A, Hutchinson S, McCulloch R, Glover L.
      Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.
  13. Cell Rep. 2021 Jan 12. pii: S2211-1247(20)31597-7. [Epub ahead of print]34(2): 108608
    Sabatella M, Thijssen KL, Davó-Martínez C, Vermeulen W, Lans H.
      Hereditary DNA repair defects affect tissues differently, suggesting that in vivo cells respond differently to DNA damage. Knowledge of the DNA damage response, however, is largely based on in vitro and cell culture studies, and it is currently unclear whether DNA repair changes depending on the cell type. Here, we use in vivo imaging of the nucleotide excision repair (NER) endonuclease ERCC-1/XPF-1 in C. elegans to demonstrate tissue-specific NER activity. In oocytes, XPF-1 functions as part of global genome NER (GG-NER) to ensure extremely rapid removal of DNA-helix-distorting lesions throughout the genome. In contrast, in post-mitotic neurons and muscles, XPF-1 participates in NER of transcribed genes only. Strikingly, muscle cells appear more resistant to the effects of DNA damage than neurons. These results suggest a tissue-specific organization of the DNA damage response and may help to better understand pleiotropic and tissue-specific consequences of accumulating DNA damage.
    Keywords:  C. elegans; CSB; DNA damage response; DNA repair; ERCC1/XPF; XPC; nucleotide excision repair; tissue specificity; transcription; transcription-coupled nucleotide excision repair
  14. NAR Cancer. 2021 Mar;3(1): zcaa037
    Lou J, Yang Y, Gu Q, Price BA, Qiu Y, Fedoriw Y, Desai S, Mose LE, Chen B, Tateishi S, Parker JS, Vaziri C, Wu D.
      The E3 ubiquitin ligase Rad18 promotes a damage-tolerant and error-prone mode of DNA replication termed trans-lesion synthesis that is pathologically activated in cancer. However, the impact of vertebrate Rad18 on cancer genomes is not known. To determine how Rad18 affects mutagenesis in vivo, we have developed and implemented a novel computational pipeline to analyze genomes of carcinogen (7, 12-Dimethylbenz[a]anthracene, DMBA)-induced skin tumors from Rad18+/+ and Rad18- / - mice. We show that Rad18 mediates specific mutational signatures characterized by high levels of A(T)>T(A) single nucleotide variations (SNVs). In Rad18- /- tumors, an alternative mutation pattern arises, which is characterized by increased numbers of deletions >4 bp. Comparison with annotated human mutational signatures shows that COSMIC signature 22 predominates in Rad18+/+ tumors whereas Rad18- / - tumors are characterized by increased contribution of COSMIC signature 3 (a hallmark of BRCA-mutant tumors). Analysis of The Cancer Genome Atlas shows that RAD18 expression is strongly associated with high SNV burdens, suggesting RAD18 also promotes mutagenesis in human cancers. Taken together, our results show Rad18 promotes mutagenesis in vivo, modulates DNA repair pathway choice in neoplastic cells, and mediates specific mutational signatures that are present in human tumors.
  15. DNA Repair (Amst). 2021 Jan 07. pii: S1568-7864(20)30296-2. [Epub ahead of print]98 103036
    Malakoti F, Alemi F, Younesi S, Majidinia M, Yousefi B, Morovat P, Khelghati N, Maleki M, Karimian A, Asemi Z.
      The DNA damage response (DDR) pathway's primary purpose is to maintain the genome structure's integrity and stability. A great deal of effort has done to understand the exact molecular mechanisms of non-coding RNAs, such as lncRNA, miRNAs, and circRNAs, in distinct cellular and genomic processes and cancer progression. In this regard, the ncRNAs possible regulatory role in DDR via modulation of key components expression and controlling repair signaling pathway activation is validated. Therefore, in this article, we will discuss the latest developments of ncRNAs contribution in different aspects of DNA repair through regulation of ATM-ATR, P53, and other regulatory signaling pathways.
    Keywords:  ATM; ATR; DNA damage; DNA repair; Noncoding RNA
  16. Biochim Biophys Acta Gen Subj. 2021 Jan 09. pii: S0304-4165(21)00001-5. [Epub ahead of print] 129842
    Raza MZ, Cadassou O, Dumontet C, Cros-Perrial E, Jordheim LP.
      BACKGROUND: Cytosolic 5'-nucleotidase II (cN-II) and ecto-5'-nucleotidase (CD73) are enzymes involved in the nucleotide metabolism by dephosphorylating nucleoside monophosphates. Both enzymes are involved in cancer by modifying anticancer drug activity, cancer cell biology and immune modulation.METHODS: We have modified lung cancer cells (NCI-H292) to become deficient for either or both enzymes using the CRISPR/Cas9 technique, and studied the implication of the two enzymes in the cellular response to different stress condition i.e. chemotherapeutic agents, hypoxia and nucleotide stress.
    RESULTS: Our results show that there is no significant role of these enzymes in cell proliferation under hypoxic stress. Similarly, cN-II and CD73 are not involved in wound healing ability under CoCl2-mediated HIF-1α stabilization. Furthermore, our results show that CD73-deficiency is associated with increased apoptosis in response to 1600 μM adenosine, decreased sensitivity to mitomycin and enhanced sensitivity to vincristine. cN-II deficiency increased in vivo tumor growth and sensitivity to vincristine and mitomycin C.
    CONCLUSIONS: Our study gives new insights into the biological roles of cN-II and CD73 under stress conditions in this particular cancer cell line. Further experiments will help deciphering the molecular mechanisms underlying the observed differences.
    Keywords:  5′-nucleotidase; Cancer cells; Cancer drugs; Cellular response; Hypoxia
  17. Nat Commun. 2021 Jan 15. 12(1): 401
    Shafi AA, McNair CM, McCann JJ, Alshalalfa M, Shostak A, Severson TM, Zhu Y, Bergman A, Gordon N, Mandigo AC, Chand SN, Gallagher P, Dylgjeri E, Laufer TS, Vasilevskaya IA, Schiewer MJ, Brunner M, Feng FY, Zwart W, Knudsen KE.
      Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.
  18. J Mol Biol. 2021 Jan 12. pii: S0022-2836(21)00005-X. [Epub ahead of print] 166811
    Raper AT, Maxwell BA, Suo Z.
      Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2'-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein-protein interactions with APE1 to ensure timely repair of damaged DNA.
    Keywords:  8-oxo-7,8-dihydro-2ʹ-deoxoguanine; Base excision repair; human 8-oxoguanine glycosylase; human AP endonuclease; single-molecule Förster resonance energy transfer; stopped-flow
  19. PLoS Genet. 2021 Jan;17(1): e1009302
    Saini N, Giacobone CK, Klimczak LJ, Papas BN, Burkholder AB, Li JL, Fargo DC, Bai R, Gerrish K, Innes CL, Schurman SH, Gordenin DA.
      Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.
  20. Mol Cell. 2021 Jan 04. pii: S1097-2765(20)30938-2. [Epub ahead of print]
    Roy U, Kwon Y, Marie L, Symington L, Sung P, Lisby M, Greene EC.
      Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
    Keywords:  DNA curtains; DNA repair; Rad51; Rad51 paralogs; Rad55-Rad57; Srs2; homologous recombination; single-molecule
  21. Nat Commun. 2021 01 14. 12(1): 366
    Tajan M, Hennequart M, Cheung EC, Zani F, Hock AK, Legrave N, Maddocks ODK, Ridgway RA, Athineos D, Suárez-Bonnet A, Ludwig RL, Novellasdemunt L, Angelis N, Li VSW, Vlachogiannis G, Valeri N, Mainolfi N, Suri V, Friedman A, Manfredi M, Blyth K, Sansom OJ, Vousden KH.
      Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.
  22. Curr Genet. 2021 Jan 11.
    Ahamad N, Khan S, Mahdi ATA, Xu YJ.
      DNA replication checkpoint is a cell signaling pathway that is activated in response to perturbed replication. Although it is crucial for maintaining genomic integrity and cell survival, the exact mechanism of the checkpoint signaling remains to be understood. Emerging evidence has shown that RecQ helicases, a large family of helicases that are conserved from bacteria to yeasts and humans, contribute to the replication checkpoint as sensors, adaptors, or regulation targets. Here, we highlight the multiple functions of RecQ helicases in the replication checkpoint in four model organisms and present additional evidence that fission yeast RecQ helicase Rqh1 may participate in the replication checkpoint as a sensor.
    Keywords:  ATR; BLM; Budding yeast; CHK1; Cds1; DNA replication checkpoint; Fission yeast; Genomic instability; Helicase; Mec1; RECQ1; RECQ4; RECQ5; Rad3; RecQ; Replication stress; Rqh1; SOS response; Sgs1; WRN
  23. NAR Cancer. 2021 Mar;3(1): zcaa044
    Liddiard K, Grimstead JW, Cleal K, Evans A, Baird DM.
      Identifying attributes that distinguish pre-malignant from senescent cells provides opportunities for targeted disease eradication and revival of anti-tumour immunity. We modelled a telomere-driven crisis in four human fibroblast lines, sampling at multiple time points to delineate genomic rearrangements and transcriptome developments that characterize the transition from dynamic proliferation into replicative crisis. Progression through crisis was associated with abundant intra-chromosomal telomere fusions with increasing asymmetry and reduced microhomology usage, suggesting shifts in DNA repair capacity. Eroded telomeres also fused with genomic loci actively engaged in transcription, with particular enrichment in long genes. Both gross copy number alterations and transcriptional responses to crisis likely underpin the elevated frequencies of telomere fusion with chromosomes 9, 16, 17, 19 and most exceptionally, chromosome 12. Juxtaposition of crisis-regulated genes with loci undergoing de novo recombination exposes the collusive contributions of cellular stress responses to the evolving cancer genome.
  24. Pharmaceutics. 2021 Jan 08. pii: E75. [Epub ahead of print]13(1):
    Escalante PI, Quiñones LA, Contreras HR.
      The FOLFOX scheme, based on the association of 5-fluorouracil and oxaliplatin, is the most frequently indicated chemotherapy scheme for patients diagnosed with metastatic colorectal cancer. Nevertheless, development of chemoresistance is one of the major challenges associated with this disease. It has been reported that epithelial-mesenchymal transition (EMT) is implicated in microRNA-driven modulation of tumor cells response to 5-fluorouracil and oxaliplatin. Moreover, from pharmacogenomic research, it is known that overexpression of genes encoding dihydropyrimidine dehydrogenase (DPYD), thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), the DNA repair enzymes ERCC1, ERCC2, and XRCC1, and the phase 2 enzyme GSTP1 impair the response to FOLFOX. It has been observed that EMT is associated with overexpression of DPYD, TYMS, ERCC1, and GSTP1. In this review, we investigated the role of miRNAs as EMT promotors in tumor cells, and its potential effect on the upregulation of DPYD, TYMS, MTHFR, ERCC1, ERCC2, XRCC1, and GSTP1 expression, which would lead to resistance of CRC tumor cells to 5-fluorouracil and oxaliplatin. This constitutes a potential mechanism of epigenetic regulation involved in late-onset of acquired resistance in mCRC patients under FOLFOX chemotherapy. Expression of these biomarker microRNAs could serve as tools for personalized medicine, and as potential therapeutic targets in the future.
    Keywords:  5-fluorouracil; EMT-transcription factors; FOLFOX; biomarker; chemoresistance; epithelial-mesenchymal transition; microRNA; oxaliplatin; pharmacoepigenetics; pharmacogenetics
  25. Nucleic Acids Res. 2021 Jan 14. pii: gkaa1297. [Epub ahead of print]
    Jurkiw TJ, Tumbale PP, Schellenberg MJ, Cunningham-Rundles C, Williams RS, O'Brien PJ.
      Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg2+ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg2+ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg2+ levels.
  26. Nucleic Acids Res. 2021 Jan 15. pii: gkaa1082. [Epub ahead of print]
    Wang L, Zhan L, Zhao Y, Huang Y, Wu C, Pan T, Qin Q, Xu Y, Deng Z, Li J, Hu H, Xue S, Yan S.
      DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.
  27. Nat Commun. 2021 01 13. 12(1): 359
    Griesbach E, Schlackow M, Marzluff WF, Proudfoot NJ.
      Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3'-end processing in the maintenance of effective DDR.
  28. Biomolecules. 2021 Jan 08. pii: E76. [Epub ahead of print]11(1):
    Lirussi L, Demir Ö, You P, Sarno A, Amaro RE, Nilsen H.
      RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.
    Keywords:  SMUG1; modified bases; rRNA processing
  29. Nat Commun. 2021 01 12. 12(1): 321
    Shen M, Dhingra N, Wang Q, Cheng C, Zhu S, Tian X, Yu J, Gong X, Li X, Zhang H, Xu X, Zhai L, Xie M, Gao Y, Deng H, He Y, Niu H, Zhao X, Xiang S.
      The yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5's activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5's HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance.
  30. Cell Host Microbe. 2021 Jan 07. pii: S1931-3128(20)30670-3. [Epub ahead of print]
    Nayak RR, Alexander M, Deshpande I, Stapleton-Gray K, Rimal B, Patterson AD, Ubeda C, Scher JU, Turnbaugh PJ.
      Immunomodulatory drugs can inhibit bacterial growth, yet their mechanism of action, spectrum, and clinical relevance remain unknown. Methotrexate (MTX), a first-line rheumatoid arthritis (RA) treatment, inhibits mammalian dihydrofolate reductase (DHFR), but whether it directly impacts gut bacteria is unclear. We show that MTX broadly alters the human gut microbiota. Drug sensitivity varied across strains, but the mechanism of action against DHFR appears conserved between mammalian and bacterial cells. RA patient microbiotas were sensitive to MTX, and changes in gut bacterial taxa and gene family abundance were distinct between responders and non-responders. Transplantation of post-treatment samples into germ-free mice given an inflammatory trigger led to reduced immune activation relative to pre-treatment controls, enabling identification of MTX-modulated bacterial taxa associated with intestinal and splenic immune cells. Thus, conservation in cellular pathways across domains of life can result in broad off-target drug effects on the human gut microbiota with consequences for immune function.
    Keywords:  DMARD; autoimmune disease; human gut microbiome; immune activation; metabolomics; methotrexate; microbiota; off-target effects; purine and pyrimidine biosynthesis; rheumatoid arthritis
  31. Bioorg Chem. 2020 Dec 23. pii: S0045-2068(20)31875-7. [Epub ahead of print]107 104577
    Ghoteimi R, Braka A, Rodriguez C, Cros-Perrial E, Tai Nguyen V, Uttaro JP, Mathé C, Chaloin L, Ménétrier-Caux C, Jordheim LP, Peyrottes S.
      Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-β-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The β-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.
    Keywords:  5′-ectonucleotidase; Bis-phosphonate; Click chemistry; Enzyme inhibitor; Immuno-oncology; Nucleotide