bims-numges 2020-08-16 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020‒08‒16
38 papers selected by
Sean Rudd
Karolinska Institutet

Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network.
XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells.
BRD4 prevents the accumulation of R-loops and protects against transcription-replication collision events and DNA damage.
FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.
FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function.
The role of α tubulin acetyltransferase αTAT1 in DNA damage response.
Replication protein A binds RNA and promotes R-loop formation.
The Chromatin Response to Double-Strand DNA Breaks and Their Repair.
Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.
γH2AX in the S Phase After UV Irradiation Corresponds to DNA Replication and Does Not Report on the Extent of DNA Damage.
DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.
Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.
Maintenance of genome integrity and active homologous recombination in embryonic stem cells.
Non-canonical ATM/MRN activities temporally define the senescence secretory program.
Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States.
A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice.
PARP Inhibitors & Prostate Cancer: To Infinity and Beyond BRCA.
Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.
Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells.
ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents.
Remarkable Enhancement of Nucleotide Excision Repair of a Bulky Guanine Lesion in a Covalently Closed Circular DNA Plasmid Relative to the Same Linearized Plasmid.
The E2F1 transcription factor and RB tumor suppressor moonlight as DNA repair factors.
ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex.
Serine restriction alters sphingolipid diversity to constrain tumour growth.
An assay for DNA polymerase β lyase inhibitors that engage the catalytic nucleophile for binding.
On the irrelevancy of hydroxyl radical to DNA damage from oxidative stress and implications for epigenetics.
Checkpoint adaptation in recombination-deficient cells drives aneuploidy and resistance to genotoxic agents.
Active turnover of genomic methylcytosine in pluripotent cells.
cGAS-STING pathway in oncogenesis and cancer therapeutics.
Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos.
Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.
Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration.
Genetic investigation of purine nucleotide imbalance in Saccharomyces cerevisiae.
Shikonin is a novel and selective IMPDH2 inhibitor that target triple-negative breast cancer.
Identification of three new compounds that directly target human serine hydroxymethyltransferase SHMT2.
Cryo-EM structure of the human concentrative nucleoside transporter CNT3.
A Timeless Tale: G4 structure recognition by the fork protection complex triggers unwinding by DDX11 helicase.

Leukemia. 2020 Aug 07.

Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network.

Xiaorong Gu, Rita Tohme, Benjamin Tomlinson, Nneha Sakre, Metis Hasipek, Lisa Durkin, Caroline Schuerger, Dale Grabowski, Asmaa M Zidan, Tomas Radivoyevitch, Changjin Hong, Hetty Carraway, Betty Hamilton, Ronald Sobecks, Bhumika Patel, Babal K Jha, Eric D Hsi, Jaroslaw Maciejewski, Yogen Saunthararajah.

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

DOI: https://doi.org/10.1038/s41375-020-1003-x
NAR Cancer. 2020 Sep;2(3): zcaa013

XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells.

Bradley J Eckelmann, Albino Bacolla, Haibo Wang, Zu Ye, Erika N Guerrero, Wei Jiang, Randa El-Zein, Muralidhar L Hegde, Alan E Tomkinson, John A Tainer, Sankar Mitra.

Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alternative DNA double-strand break repair pathways, particularly alternative non-homologous end joining or microhomology-mediated end joining (alt-EJ/MMEJ) during S and G2 cell cycle phases. Depletion of alt-EJ factors, including XRCC1, PARP1 and POLQ, is synthetically lethal with BRCA2 deficiency; yet, XRCC1 roles in HR-deficient cancers and replication stress are enigmatic. Here, we show that after replication stress, XRCC1 forms an active repair complex with POLQ and MRE11 that supports alt-EJ activity in vitro. BRCA2 limits XRCC1 recruitment and repair complex formation to suppress alt-EJ at stalled forks. Without BRCA2 fork protection, XRCC1 enables cells to complete DNA replication at the expense of increased genome instability by promoting MRE11-dependent fork resection and restart. High XRCC1 and MRE11 gene expression negatively impacts Kaplan-Meier survival curves and hazard ratios for HR-deficient breast cancer patients in The Cancer Genome Atlas. The additive effects of depleting both BRCA2 and XRCC1 indicate distinct pathways for replication restart. Our collective data show that XRCC1-mediated processing contributes to replication fork degradation, replication restart and chromosome aberrations in BRCA2-deficient cells, uncovering new roles of XRCC1 and microhomology-mediated repair mechanisms in HR-deficient cancers, with implications for chemotherapeutic strategies targeting POLQ and PARP activities.

DOI: https://doi.org/10.1093/narcan/zcaa013
Nat Commun. 2020 Aug 14. 11(1): 4083

BRD4 prevents the accumulation of R-loops and protects against transcription-replication collision events and DNA damage.

Fred C Lam, Yi Wen Kong, Qiuying Huang, Tu-Lan Vu Han, Amanda D Maffa, Ekkehard M Kasper, Michael B Yaffe.

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.

DOI: https://doi.org/10.1038/s41467-020-17503-y
Nucleic Acids Res. 2020 Aug 14. pii: gkaa660. [Epub ahead of print]

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

Sanket Awate, Joshua A Sommers, Arindam Datta, Sumeet Nayak, Marina A Bellani, Olivia Yang, Christopher A Dunn, Claudia M Nicolae, George-Lucian Moldovan, Michael M Seidman, Sharon B Cantor, Robert M Brosh.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

DOI: https://doi.org/10.1093/nar/gkaa660
Cell Rep. 2020 Aug 11. pii: S2211-1247(20)31003-2. [Epub ahead of print]32(6): 108018

FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function.

Mengfan Tang, Xu Feng, Guangsheng Pei, Mrinal Srivastava, Chao Wang, Zhen Chen, Siting Li, Huimin Zhang, Zhongming Zhao, Xu Li, Junjie Chen.

53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1-53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.

DOI: https://doi.org/10.1016/j.celrep.2020.108018
J Cell Sci. 2020 Aug 11. pii: jcs.246702. [Epub ahead of print]

The role of α tubulin acetyltransferase αTAT1 in DNA damage response.

Na Mi Ryu, Jung Min Kim.

  Lysine 40 acetylation of α tubulin (Ac-α tubulin) catalyzed by acetyltransferase αTAT1, marks stabilized microtubules. Recently, there is growing evidence to suggest the crosstalk between DNA damage response (DDR) and microtubule organization, we therefore investigated whether αTAT1 is involved in DDR. Following treatment with DNA damaging agents, increased levels of Ac-α tubulin were detected. We also observed significant induction of Ac-α tubulin after depletion of DNA repair proteins, suggesting that αTAT1 is positively regulated in response to DNA damage. Intriguingly, αTAT1 depletion decreased DNA damage-induced RPA phosphorylation and foci formation. Moreover, DNA damage -induced cell cycle arrest was significantly delayed in αTAT1-depleted cells, indicating defective checkpoint activation. The checkpoint defects by αTAT1 deficiency were restored by expression of wild-type αTAT1, but not by D157N αTAT1 (catalytically inactive αTAT1), indicating that the role of αTAT1 in the DDR is dependent on enzymatic activity. Furthermore, αTAT1-depleted DR-GFP U2OS cells resulted in a significant decrease in the frequency of homologous recombination repair. Collectively, our results suggest that αTAT1 may play an essential role in DNA damage checkpoints and DNA repair through its acetyltransferase activity.

Keywords:  Checkpoint activation; DNA damage response; Homologous recombination repair; Microtubule stability; α tubulin acetyltransferase αTAT1; α tubulin lysine (K40) acetylation

DOI:  https://doi.org/10.1242/jcs.246702
J Biol Chem. 2020 Aug 12. pii: jbc.RA120.013812. [Epub ahead of print]

Replication protein A binds RNA and promotes R-loop formation.

Olga M Mazina, Srinivas Somarowthu, Lyudmila Y Kadyrova, Andrey G Baranovskiy, Tahir H Tahirov, Farid A Kadyrov, Alexander V Mazin.

  Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes including humans and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays we found that RPA binds RNA with an unexpectedly high affinity (KD ≈ 100 pM). Furthermore, RPA by forming a complex with RNA can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis mimicking the process of replication restart in vivo. These results demonstrate that RPA binds RNA with high affinity supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.

Keywords:  DNA polymerase; DNA repair; DNA replication; DNA replication restart; R-loop extension; R-loops; RNA; RNA binding protein; homologous recombination

DOI:  https://doi.org/10.1074/jbc.RA120.013812
Cells. 2020 Aug 07. pii: E1853. [Epub ahead of print]9(8):

The Chromatin Response to Double-Strand DNA Breaks and Their Repair.

Radoslav Aleksandrov, Rossitsa Hristova, Stoyno Stoynov, Anastas Gospodinov.

  Cellular DNA is constantly being damaged by numerous internal and external mutagenic factors. Probably the most severe type of insults DNA could suffer are the double-strand DNA breaks (DSBs). They sever both DNA strands and compromise genomic stability, causing deleterious chromosomal aberrations that are implicated in numerous maladies, including cancer. Not surprisingly, cells have evolved several DSB repair pathways encompassing hundreds of different DNA repair proteins to cope with this challenge. In eukaryotic cells, DSB repair is fulfilled in the immensely complex environment of the chromatin. The chromatin is not just a passive background that accommodates the multitude of DNA repair proteins, but it is a highly dynamic and active participant in the repair process. Chromatin alterations, such as changing patterns of histone modifications shaped by numerous histone-modifying enzymes and chromatin remodeling, are pivotal for proficient DSB repair. Dynamic chromatin changes ensure accessibility to the damaged region, recruit DNA repair proteins, and regulate their association and activity, contributing to DSB repair pathway choice and coordination. Given the paramount importance of DSB repair in tumorigenesis and cancer progression, DSB repair has turned into an attractive target for the development of novel anticancer therapies, some of which have already entered the clinic.

Keywords:  DNA damage response; PARP inhibitors; anticancer drug therapies; cancer; chromatin dynamics in DNA repair; double-strand DNA break repair; homologous recombination; non-homologous end joining; synthetic lethality

DOI:  https://doi.org/10.3390/cells9081853
Am J Physiol Cell Physiol. 2020 Aug 12.

Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.

Rongyi Shi, Yiyi Wang, Ya Gao, Xiaoli Xu, Shuyu Mao, Yue Xiao, Shuang Song, Liangyan Wang, Bing Tian, Ye Zhao, Yuejin Hua, Hong Xu.

  Human flap endonuclease 1 (FEN1) is a structure-specific, multi-functional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as UV irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintain genome stability.

Keywords:  DNA repair; Flap endonuclease 1; lysine acetylation; lysine succinylation; posttranslational modification

DOI:  https://doi.org/10.1152/ajpcell.00137.2020
Mol Cell Biol. 2020 Aug 10. pii: MCB.00328-20. [Epub ahead of print]

γH2AX in the S Phase After UV Irradiation Corresponds to DNA Replication and Does Not Report on the Extent of DNA Damage.

Shivnarayan Dhuppar, Sitara Roy, Aprotim Mazumder.

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX - the phosphorylated form of a histone variant H2AX - in the S phase within an asynchronous population of cells. γH2AX is often considered as a definitive marker of DNA damage inside a cell. In this report we show that γH2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double strand breaks or in the inhibition of global transcription. Instead γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of γH2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage but the response elicited which peaks in the S phase upon UV irradiation.

DOI: https://doi.org/10.1128/MCB.00328-20
Nat Commun. 2020 Aug 07. 11(1): 3951

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain.

Jing Zhang, Marina A Bellani, Ryan C James, Durga Pokharel, Yongqing Zhang, John J Reynolds, Gavin S McNee, Andrew P Jackson, Grant S Stewart, Michael M Seidman.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

DOI: https://doi.org/10.1038/s41467-020-17449-1
EMBO J. 2020 Aug 13. e105705

Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.

J Brooks Crickard, Youngho Kwon, Patrick Sung, Eric C Greene.

  Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.

Keywords:  DNA repair; Rad51; Rad54; Rdh54; homologous recombination

DOI:  https://doi.org/10.15252/embj.2020105705
J Radiat Res. 2020 Aug 11. pii: rraa053. [Epub ahead of print]

Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.

Atsushi Shibata, Penny A Jeggo.

  p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1's breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

Keywords:  53BP1; DNA double-strand break repair; DNA-PK; homologous recombination; ionising radiation; non-homologous end-joining

DOI:  https://doi.org/10.1093/jrr/rraa053
Exp Mol Med. 2020 Aug 07.

Maintenance of genome integrity and active homologous recombination in embryonic stem cells.

Eui-Hwan Choi, Seobin Yoon, Young Eun Koh, Young-Jin Seo, Keun Pil Kim.

Embryonic stem cells (ESCs) possess specific gene expression patterns that confer the ability to proliferate indefinitely and enable pluripotency, which allows ESCs to differentiate into diverse cell types in response to developmental signals. Compared to differentiated cells, ESCs harbor an elevated level of homologous recombination (HR)-related proteins and exhibit exceptional cell cycle control, characterized by a high proliferation rate and a prolonged S phase. HR is involved in several aspects of chromosome maintenance. For instance, HR repairs impaired chromosomes and prevents the collapse of DNA replication forks during cell proliferation. Thus, HR is essential for the maintenance of genomic integrity and prevents cellular dysregulation and lethal events. In addition, abundant HR proteins in the prolonged S phase can efficiently protect ESCs from external damages and protect against genomic instability caused by DNA breaks, facilitating rapid and accurate DNA break repair following chromosome duplication. The maintenance of genome integrity is key to preserving the functions of ESCs and reducing the risks of cancer development, cell cycle arrest, and abnormal replication. Here, we review the fundamental links between the stem cell-specific HR process and DNA damage response as well as the different strategies employed by ESCs to maintain genomic integrity.

DOI: https://doi.org/10.1038/s12276-020-0481-2
EMBO Rep. 2020 Aug 12. e50718

Non-canonical ATM/MRN activities temporally define the senescence secretory program.

Nicolas Malaquin, Marc-Alexandre Olivier, Aurélie Martinez, Stéphanie Nadeau, Christina Sawchyn, Jean-Philippe Coppé, Guillaume Cardin, Frédérick A Mallette, Judith Campisi, Francis Rodier.

  Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.

Keywords:  DNA damage response; MRN complex; NF-κB; chromatin; senescence secretome

DOI:  https://doi.org/10.15252/embr.202050718
Cells. 2020 Aug 10. pii: E1870. [Epub ahead of print]9(8):

Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States.

Sara Oster, Rami I Aqeilan.

  DNA double strand breaks (DSBs) are known to be the most toxic and threatening of the various types of breaks that may occur to the DNA. However, growing evidence continuously sheds light on the regulatory roles of programmed DSBs. Emerging studies demonstrate the roles of DSBs in processes such as T and B cell development, meiosis, transcription and replication. A significant recent progress in the last few years has contributed to our advanced knowledge regarding the functions of DSBs is the development of many next generation sequencing (NGS) methods, which have considerably advanced our capabilities. Other studies have focused on the implications of programmed DSBs on chromosomal aberrations and tumorigenesis. This review aims to summarize what is known about DNA damage in its physiological context. In addition, we will examine the advancements of the past several years, which have made an impact on the study of genome landscape and its organization.

Keywords:  BCR; DNA repair; NGS; chromosomal translocations; meiosis; physiological DSBs; transcription

DOI:  https://doi.org/10.3390/cells9081870
Nat Commun. 2020 Aug 14. 11(1): 4077

A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice.

Paris Roidos, Stephanie Sungalee, Salvatore Benfatto, Özdemirhan Serçin, Adrian M Stütz, Amir Abdollahi, Jan Mauer, Frank T Zenke, Jan O Korbel, Balca R Mardin.

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.

DOI: https://doi.org/10.1038/s41467-020-17962-3
Oncologist. 2020 Aug 13.

PARP Inhibitors & Prostate Cancer: To Infinity and Beyond BRCA.

Emily N Risdon, Cindy H Chau, Douglas K Price, Oliver Sartor, William D Figg.

  The U.S. Food and Drug Administration (FDA) recently approved two poly-ADP ribose polymerase (PARP) inhibitors, olaparib and rucaparib, for treatment of biomarker-positive metastatic castrate resistant prostate cancer. The benefits of PARP inhibition have been well characterized in patients that have BRCA1 and BRCA2 mutations in several forms of cancer. BRCA1 and BRCA2 occupy key roles in DNA damage repair, which is comprised of several different pathways with numerous participants. Patients with mutations in other key genes within the DNA damage repair pathway may also respond to treatment with PARP inhibitors, and identification of these alterations could significantly increase the percentage of patients that may benefit from PARP inhibition. This review focuses on the potential for synthetically lethal interactions between PARP inhibitors and non-BRCA DNA damage repair genes. IMPLICATIONS FOR PRACTICE: The treatment potential of PARP inhibition has been well characterized in patients with BRCA1 and BRCA2 mutations, but there is compelling evidence for expanding the use of PARP inhibitors to mutations of other non-BRCA DNA damage repair (DDR) genes. This could increase the percentage of patients that may benefit from treatment with PARP inhibitors alone or in combination with other therapies. Understanding the significance of PARP inhibitor-sensitizing alterations in other common non-BRCA DDR will help guide clinical decisions to provide targeted treatment options to a wider population of patients.

Keywords:  BRCA; Biomarker; DNA Damage Repair; Homologous Recombination Repair; Mismatch Repair; Mutation; Olaparib; Poly(ADP-ribose) Polymerase Inhibitor; Prostate Cancer; Rucaparib

DOI:  https://doi.org/10.1634/theoncologist.2020-0697
Nat Commun. 2020 Aug 07. 11(1): 3940

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

Alexy Promonet, Ismaël Padioleau, Yaqun Liu, Lionel Sanz, Anna Biernacka, Anne-Lyne Schmitz, Magdalena Skrzypczak, Amélie Sarrazin, Clément Mettling, Maga Rowicka, Krzysztof Ginalski, Frédéric Chedin, Chun-Long Chen, Yea-Lih Lin, Philippe Pasero.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

DOI: https://doi.org/10.1038/s41467-020-17858-2
Sci Adv. 2020 Jul;6(31): eaay9131

Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells.

Seung Ho Shin, Ji Su Lee, Jia-Min Zhang, Sungbin Choi, Zarko V Boskovic, Ran Zhao, Mengqiu Song, Rui Wang, Jie Tian, Mee-Hyun Lee, Jae Hwan Kim, Minju Jeong, Jung Hyun Lee, Michael Petukhov, Sam W Lee, Sang Gyun Kim, Lee Zou, Sanguine Byun.

Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.

DOI: https://doi.org/10.1126/sciadv.aay9131
JCI Insight. 2020 Aug 11. pii: 138117. [Epub ahead of print]

ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents.

Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abian, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, Jose Luis Neira, Juan L Iovanna.

  Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.

Keywords:  Cancer; Cell Biology; Cell stress; Oncology

DOI:  https://doi.org/10.1172/jci.insight.138117
Biochemistry. 2020 Aug 11. 59(31): 2842-2848

Remarkable Enhancement of Nucleotide Excision Repair of a Bulky Guanine Lesion in a Covalently Closed Circular DNA Plasmid Relative to the Same Linearized Plasmid.

Marina Kolbanovskiy, Abraham Aharonoff, Ana Helena Sales, Nicholas E Geacintov, Vladimir Shafirovich.

The excision of DNA lesions by human nucleotide excision repair (NER) has been extensively studied in human cell extracts. Employing DNA duplexes with fewer than 200 bp containing a single bulky, benzo[a]pyrene-derived guanine lesion (B[a]P-dG), the NER yields are typically on the order of ∼5-10%, or less. Remarkably, the NER yield is enhanced by a factor of ∼6 when the B[a]P-dG lesion is embedded in a covalently closed circular pUC19NN plasmid (contour length of 2686 bp) rather than in the same plasmid linearized by a restriction enzyme with the B[a]P-dG adduct positioned at the 945th nucleotide counted from the 5'-end of the linearized DNA molecules. Furthermore, the NER yield in the circular pUC19NN plasmid is ∼9 times greater than in a short 147-mer DNA duplex with the B[a]P-dG adduct positioned in the middle. Although the NER factors responsible for these differences were not explicitly identified here, we hypothesize that the initial DNA damage sensor XPC-RAD23B is a likely candidate; it is known to search for DNA lesions by a constrained one-dimensional search mechanism [Cheon, N. Y., et al. (2019) Nucleic Acids Res. 47, 8337-8347], and our results are consistent with the notion that it dissociates more readily from the blunt ends than from the inner regions of linear DNA duplexes, thus accounting for the remarkable enhancement in NER yields associated with the single B[a]P-dG adduct embedded in covalently closed circular plasmids.

DOI: https://doi.org/10.1021/acs.biochem.0c00441
Cell Cycle. 2020 Aug 13. 1-10

The E2F1 transcription factor and RB tumor suppressor moonlight as DNA repair factors.

Swarnalatha Manickavinayaham, Renier Velez-Cruz, Anup K Biswas, Jie Chen, Ruifeng Guo, David G Johnson.

  The E2F1 transcription factor and RB tumor suppressor are best known for their roles in regulating the expression of genes important for cell cycle progression but, they also have transcription-independent functions that facilitate DNA repair at sites of damage. Depending on the type of DNA damage, E2F1 can recruit either the GCN5 or p300/CBP histone acetyltransferases to deposit different histone acetylation marks in flanking chromatin. At DNA double-strand breaks, E2F1 also recruits RB and the BRG1 ATPase to remodel chromatin and promote loading of the MRE11-RAD50-NBS1 complex. Knock-in mouse models demonstrate important roles for E2F1 post-translational modifications in regulating DNA repair and physiological responses to DNA damage. This review highlights how E2F1 moonlights in DNA repair, thus revealing E2F1 as a versatile protein that recruits many of the same chromatin-modifying enzymes to sites of DNA damage to promote repair that it recruits to gene promoters to regulate transcription.

Keywords:  ATM/ATR; BRG1; E2F1; GCN5; TopBP1; p300/CBP

DOI:  https://doi.org/10.1080/15384101.2020.1801190
Nat Commun. 2020 Aug 13. 11(1): 4046

ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex.

Peter S J Bailey, Brian M Ortmann, Anthony W Martinelli, Jack W Houghton, Ana S H Costa, Stephen P Burr, Robin Antrobus, Christian Frezza, James A Nathan.

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.

DOI: https://doi.org/10.1038/s41467-020-17862-6
Nature. 2020 Aug 12.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Thangaselvam Muthusamy, Thekla Cordes, Michal K Handzlik, Le You, Esther W Lim, Jivani Gengatharan, Antonio F M Pinto, Mehmet G Badur, Matthew J Kolar, Martina Wallace, Alan Saghatelian, Christian M Metallo.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

DOI: https://doi.org/10.1038/s41586-020-2609-x
Bioorg Med Chem. 2020 Sep 01. pii: S0968-0896(20)30472-7. [Epub ahead of print]28(17): 115642

An assay for DNA polymerase β lyase inhibitors that engage the catalytic nucleophile for binding.

Sasha M Daskalova, Brian M Eisenhauer, Mingxuan Gao, Xizhi Feng, Xun Ji, Qi Cheng, NourEddine Fahmi, Omar M Khdour, Shengxi Chen, Sidney M Hecht.

  DNA polymerase β (Pol β) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol β thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase β (Pol β) and Pol β modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

Keywords:  Active site interaction; Enzyme modification; Lyase inhibitor; Non-proteinogenic amino acid; Targeted inhibitor selection

DOI:  https://doi.org/10.1016/j.bmc.2020.115642
Chem Soc Rev. 2020 Aug 12.

On the irrelevancy of hydroxyl radical to DNA damage from oxidative stress and implications for epigenetics.

Aaron M Fleming, Cynthia J Burrows.

Contrary to frequent reports in the literature, hydroxyl radical is not a key species participating in endogenous oxidative DNA damage. Instead, carbonate radical anion is formed from the Fenton reaction under cellular conditions and from decomposition of nitrosoperoxycarbonate generated during inflammation. Carbonate radical anion is a potent one-electron oxidant capable of generating base radical cations that can migrate over long distances in duplex DNA, ultimately generating 8-oxo-7,8-dihydroguanine at a redox-sensitive sequence such as GGG. Such a mechanism enables G-quadruplex-forming sequences to act as long-range sensors of oxidative stress, impacting gene expression via the DNA repair mechanism that reads and ultimately erases the oxidized base. With a writing, reading and erasing mechanism in place, oxidative 'damage' to DNA might be relabeled as 'epigenetic' modifications.

DOI: https://doi.org/10.1039/d0cs00579g
DNA Repair (Amst). 2020 Jul 30. pii: S1568-7864(20)30188-9. [Epub ahead of print]95 102939

Checkpoint adaptation in recombination-deficient cells drives aneuploidy and resistance to genotoxic agents.

Olga Vydzhak, Katharina Bender, Julia Klermund, Anke Busch, Stefanie Reimann, Brian Luke.

  Human cancers frequently harbour mutations in DNA repair genes, rendering the use of DNA damaging agents as an effective therapeutic intervention. As therapy-resistant cells often arise, it is important to better understand the molecular pathways that drive resistance in order to facilitate the eventual targeting of such processes. We employ recombination-defective diploid yeast as a model to demonstrate that, in response to genotoxic challenges, nearly all cells eventually undergo checkpoint adaptation, resulting in the generation of aneuploid cells with whole chromosome losses that have acquired resistance to the initial genotoxic challenge. We demonstrate that adaptation inhibition, either pharmacologically, or genetically, drastically reduces the occurrence of resistant cells. Additionally, the aneuploid phenotypes of the resistant cells can be specifically targeted to induce cytotoxicity. We provide evidence that TORC1 inhibition with rapamycin, in combination with DNA damaging agents, can prevent both checkpoint adaptation and the continued growth of aneuploid resistant cells.

Keywords:  Aneuploidy; Checkpoint adaptation; DNA repair; Genome instability; Rapamycin; Resistance

DOI:  https://doi.org/10.1016/j.dnarep.2020.102939
Nat Chem Biol. 2020 Aug 10.

Active turnover of genomic methylcytosine in pluripotent cells.

Fabio Spada, Sarah Schiffers, Angie Kirchner, Yingqian Zhang, Gautier Arista, Olesea Kosmatchev, Eva Korytiakova, René Rahimoff, Charlotte Ebert, Thomas Carell.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

DOI: https://doi.org/10.1038/s41589-020-0621-y
Oncotarget. 2020 Jul 28. 11(30): 2930-2955

cGAS-STING pathway in oncogenesis and cancer therapeutics.

Brandon Yi Da Hoong, Yunn Hwen Gan, Haiyan Liu, Ee Sin Chen.

  The host innate immunity offers the first line of defense against infection. However, recent evidence shows that the host innate immunity is also critical in sensing the presence of cytoplasmic DNA derived from genomic instability events, such as DNA damage and defective cell cycle progression. This is achieved through the cyclic GMP-AMP synthase (cGAS)/Stimulator of interferon (IFN) genes (STING) pathway. Here we discuss recent insights into the regulation of this pathway in cancer immunosurveillance, and the downstream signaling cascades that coordinate immune cell recruitment to the tumor microenvironment to destroy transformed cells through cellular senescence or cell death programs. Its central role in immunosurveillance positions the cGAS-STING pathway as an attractive anti-cancer immunotherapeutic drug target for chemical agonists or vaccine adjuvants and suggests a key node to be targeted in a synthetic lethal approach. We also discuss adaptive mechanisms used by cancer cells to circumvent cGAS-STING signaling and present evidence linking chronic cGAS-STING activation to inflammation-induced carcinogenesis, cautioning against the use of activating the cGAS-STING pathway as an anti-tumor immunotherapy. A deeper mechanistic understanding of the cGAS-STING pathway will aid in the identification of potentially efficacious anti-cancer therapeutic targets.

Keywords:  STING; cGAS; cyclic GMP-AMP synthase; interferon; stimulator of interferon

DOI:  https://doi.org/10.18632/oncotarget.27673
Nat Metab. 2020 Aug 10.

Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos.

Sushma Sharma, Camilla Koolmeister, Phong Tran, Anna Karin Nilsson, Nils-Göran Larsson, Andrei Chabes.

DOI: https://doi.org/10.1038/s42255-020-0264-z
Nat Chem Biol. 2020 Aug 10.

Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.

Mariluz Soula, Ross A Weber, Omkar Zilka, Hanan Alwaseem, Konnor La, Frederick Yen, Henrik Molina, Javier Garcia-Bermudez, Derek A Pratt, Kıvanç Birsoy.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.

DOI: https://doi.org/10.1038/s41589-020-0613-y
Cell Death Differ. 2020 Aug 07.

Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration.

Zhaoying Yang, Xiaocui Zhao, Weina Shang, Yang Liu, Jun-Feng Ji, Jun-Ping Liu, Chao Tong.

Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few β-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.

DOI: https://doi.org/10.1038/s41418-020-0601-5
Curr Genet. 2020 Aug 11.

Genetic investigation of purine nucleotide imbalance in Saccharomyces cerevisiae.

Christelle Saint-Marc, Johanna Ceschin, Claire Almyre, Benoît Pinson, Bertrand Daignan-Fornier.

  Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved.

Keywords:  AMP deaminase; Adenine deaminase; Amino acid transport; GTP; Yeast metabolism

DOI:  https://doi.org/10.1007/s00294-020-01101-y
Phytother Res. 2020 Aug 10.

Shikonin is a novel and selective IMPDH2 inhibitor that target triple-negative breast cancer.

Wanyan Wang, Yu Wu, Si Chen, Xi Liu, Jiacheng He, Shuyi Wang, Weiqiang Lu, Yong Tang, Jin Huang.

  Triple-negative breast cancer (TNBC) is heterogeneous disease with a poor prognosis. It is therefore important to explore novel therapeutic agents to improve the clinical efficacy for TNBC. The inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is always overexpressed in many types of tumors, including TNBC and regarded as a potential target for cancer therapy. Through screening a library of natural products, we identified shikonin, a natural bioactive component of Lithospermum erythrorhizon, is a novel and selective IMPDH2 inhibitor. Enzymatic analysis using Lineweaver-Burk plot indicates that shikonin is a competitive inhibitor of IMPDH2. The interaction between shikonin and IMDPH2 was further investigated by thermal shift assay, fluorescence quenching, and molecular docking simulation. Shikonin treatment effectively inhibits the growth of human TNBC cell line MDA-MB-231, and murine TNBC cell line, 4T1 in a dose-dependent manner, which is impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. Most importantly, IMPDH2 knockdown significantly reduced cell proliferation and conferred resistance to shikonin in TNBC. Collectively, our findings showed the natural product shikonin as a selective inhibitor of IMPDH2 with anti-TNBC activity, impelling its further study in clinical trials.

Keywords:  IMPDH2; guanine nucleotides; natural product; shikonin; triple-negative breast cancer

DOI:  https://doi.org/10.1002/ptr.6825
Chem Biol Drug Des. 2020 Aug 11.

Identification of three new compounds that directly target human serine hydroxymethyltransferase SHMT2.

Yanfang Han, Liping He, Yifei Qi, Yue Zhao, Yue Pan, Bohuan Fang, Sha Li, John Z H Zhang, Lujia Zhang.

  Mitochondrial serine hydroxymethyltransferase 2 (SHMT2) is an important drug target in the one-carbon metabolic pathway, since its activity is critical for purine and pyrimidine biosynthesis. Additionally, it plays a prominent role during metabolic reprogramming of cancer cells, and SHMT2 inhibitors have proven useful as anticancer drugs. Compared to drugs targeting one-carbon metabolic enzymes (mainly dihydrofolate reductase and thymidylate synthase) that have been used for clinical treatment of cancer, efficient SHMT2-specific inhibitors are lacking. Therefore, we established a direct system for virtual screening, protein expression, and identification of inhibitors targeting SHMT2. First, 27 compounds qualifying as potential SHMT2 inhibitors were selected for biological activity verification through virtual screening of the 210 thousand compounds registered in the Specs database. Second, these 27 hits were subjected to quick screening by an in vitro non-competitive kinetic assay of SHMT2 single-enzyme catalysis. This allowed us to identify three compounds featuring medium-strength and non-competitive inhibition of SHMT2: AM-807/42004511 (IC50 = 14.52 ± 4.1665 μM), AM-807/40675298 (IC50 = 12.74 ± 5.8991 μM), and AM-807/42004633 (IC50 = 9.43 ± 0.5646 μM). We describe a quick screening method for the identification of inhibitors targeting SHMT2, providing a basis for subsequent identification and screening of new inhibitors.

Keywords:  SHMT2; inhibitors; non-competitive; one-carbon metabolism; virtual screening

DOI:  https://doi.org/10.1111/cbdd.13774
PLoS Biol. 2020 Aug 10. 18(8): e3000790

Cryo-EM structure of the human concentrative nucleoside transporter CNT3.

Yanxia Zhou, Lianghuan Liao, Chen Wang, Jialu Li, Pengliang Chi, Qingjie Xiao, Qingting Liu, Li Guo, Linfeng Sun, Dong Deng.

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.

DOI: https://doi.org/10.1371/journal.pbio.3000790
EMBO J. 2020 Aug 13. e106305

A Timeless Tale: G4 structure recognition by the fork protection complex triggers unwinding by DDX11 helicase.

Catherine H Freudenreich.

How the replisome senses and deals with DNA secondary structures has been a mystery. A new study from the Sale and Pellegrini laboratories finds that the Timeless protein has a G-quadruplex binding domain that works together with the DDX11 helicase to facilitate replication of G4 DNA structures.

DOI: https://doi.org/10.15252/embj.2020106305