bims-numges 2021-07-18 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒07‒18
27 papers selected by
Sean Rudd
Karolinska Institutet

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity.
p53 deficiency induces MTHFD2 transcription to promote cell proliferation and restrain DNA damage.
SI-MOIRAI: A new method to identify and quantify the metabolic fate of nucleotides.
Selective therapeutic strategy for p53-deficient cancer by targeting dysregulation in DNA repair.
The Replication Stress Response on a Narrow Path Between Genomic Instability and Inflammation.
Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage.
Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA.
Sp1-dependent recruitment of the histone acetylase p300 to DSBs facilitates chromatin remodeling and recruitment of the NHEJ repair factor Ku70.
Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.
DNA helicases in homologous recombination repair.
CtBP1/2 differentially regulate genomic stability and DNA repair pathway in high-grade serous ovarian cancer cell.
Role of BRCA2 DNA-binding and C-terminal domain on its mobility and conformation in DNA repair.
Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.
The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair.
Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.
Repositioning PARP inhibitors in the treatment of thoracic malignancies.
CRIP1 cooperates with BRCA2 to drive the nuclear enrichment of RAD51 and to facilitate homologous repair upon DNA damage induced by chemotherapy.
NONO phase separation enhances DNA damage repair by accelerating nuclear EGFR-induced DNA-PK activation.
MEKK1-dependent activation of the CRL4 complex is important for DNA damage-induced degradation of p21 and DDB2 and cell survival.
Stochastic initiation of DNA replication across the human genome.
A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.
The intersection of DNA replication with antisense 3' RNA processing in Arabidopsis FLC chromatin silencing.
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans.
The mechanism of gap creation by a multifunctional nuclease during base excision repair.
mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis.
Pterostilbene inhibits hepatocellular carcinoma proliferation and HBV replication by targeting ribonucleotide reductase M2 protein.
FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cell Chem Biol. 2021 Jul 13. pii: S2451-9456(21)00306-8. [Epub ahead of print]

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity.

Ying Yu Liang, Smaranda Bacanu, Lekshmy Sreekumar, Anderson Daniel Ramos, Lingyun Dai, Martin Michaelis, Jindrich Cinatl, Takahiro Seki, Yihai Cao, Cynthia R Coffill, David P Lane, Nayana Prabhu, Pär Nordlund.

  The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.

Keywords:  5-fluorouracil; CETSA; cancer; cellular thermal shift assay; drug mechanism of action; drug resistance; proteomics

DOI:  https://doi.org/10.1016/j.chembiol.2021.06.007
Proc Natl Acad Sci U S A. 2021 Jul 13. pii: e2019822118. [Epub ahead of print]118(28):

p53 deficiency induces MTHFD2 transcription to promote cell proliferation and restrain DNA damage.

Gen Li, Jun Wu, Le Li, Peng Jiang.

  Cancer cells acquire metabolic reprogramming to satisfy their high biogenetic demands, but little is known about how metabolic remodeling enables cancer cells to survive stress associated with genomic instability. Here, we show that the mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) is transcriptionally suppressed by p53, and its up-regulation by p53 inactivation leads to increased folate metabolism, de novo purine synthesis, and tumor growth in vivo and in vitro. Moreover, MTHFD2 unexpectedly promotes nonhomologous end joining in response to DNA damage by forming a complex with PARP3 to enhance its ribosylation, and the introduction of a PARP3-binding but enzymatically inactive MTHFD2 mutant (e.g., D155A) sufficiently prevents DNA damage. Notably, MTHFD2 depletion strongly restrains p53-deficient cell proliferation and sensitizes cells to chemotherapeutic agents, indicating a potential role for MTHFD2 depletion in the treatment of p53-deficient tumors.

Keywords:  MTHFD2; NHEJ; cell proliferation; folate metabolism; p53

DOI:  https://doi.org/10.1073/pnas.2019822118
J Biochem. 2021 Jul 09. pii: mvab077. [Epub ahead of print]

SI-MOIRAI: A new method to identify and quantify the metabolic fate of nucleotides.

Yoshiki Ikeda, Akiyoshi Hirayama, Satoshi Kofuji, Yoshihisa Hirota, Ryo Kamata, Natsuki Osaka, Yuki Fujii, Mika Sasaki, Satsuki Ikeda, Eric P Smith, Robert Bachoo, Tomoyoshi Soga, Atsuo T Sasaki.

  Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health, and disease. However, there remains no established method to investigate quantitively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labeling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma U87MG cells and a patient-derived xenograft (PDX) glioblastoma mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in glioblastoma (GBM) in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.

Keywords:  cancer metabolism; flux analysis; glioblastoma (GBM); mass spectrometry; metabolomics; nucleotide metabolism

DOI:  https://doi.org/10.1093/jb/mvab077
Commun Biol. 2021 Jul 12. 4(1): 862

Selective therapeutic strategy for p53-deficient cancer by targeting dysregulation in DNA repair.

Justin Zonneville, Moyi Wang, Mohammed M Alruwaili, Brandon Smith, Megan Melnick, Kevin H Eng, Thomas Melendy, Ben Ho Park, Renuka Iyer, Christos Fountzilas, Andrei V Bakin.

Breast carcinomas commonly carry mutations in the tumor suppressor p53, although therapeutic efforts to target mutant p53 have previously been unfruitful. Here we report a selective combination therapy strategy for treatment of p53 mutant cancers. Genomic data revealed that p53 mutant cancers exhibit high replication activity and express high levels of the Base-Excision Repair (BER) pathway, whereas experimental testing showed substantial dysregulation in BER. This defect rendered accumulation of DNA damage in p53 mutant cells upon treatment with deoxyuridine analogues. Notably, inhibition of poly (ADP-ribose) polymerase (PARP) greatly enhanced this response, whereas normal cells responded with activation of the p53-p21 axis and cell cycle arrest. Inactivation of either p53 or p21/CDKN1A conferred the p53 mutant phenotype. Preclinical animal studies demonstrated a greater anti-neoplastic efficacy of the drug combination (deoxyuridine analogue and PARP inhibitor) than either drug alone. This work illustrates a selective combination therapy strategy for p53 mutant cancers that will improve survival rates and outcomes for thousands of breast cancer patients.

DOI: https://doi.org/10.1038/s42003-021-02370-0
Front Cell Dev Biol. 2021 ;9 702584

The Replication Stress Response on a Narrow Path Between Genomic Instability and Inflammation.

Hervé Técher, Philippe Pasero.

  The genome of eukaryotic cells is particularly at risk during the S phase of the cell cycle, when megabases of chromosomal DNA are unwound to generate two identical copies of the genome. This daunting task is executed by thousands of micro-machines called replisomes, acting at fragile structures called replication forks. The correct execution of this replication program depends on the coordinated action of hundreds of different enzymes, from the licensing of replication origins to the termination of DNA replication. This review focuses on the mechanisms that ensure the completion of DNA replication under challenging conditions of endogenous or exogenous origin. It also covers new findings connecting the processing of stalled forks to the release of small DNA fragments into the cytoplasm, activating the cGAS-STING pathway. DNA damage and fork repair comes therefore at a price, which is the activation of an inflammatory response that has both positive and negative impacts on the fate of stressed cells. These new findings have broad implications for the etiology of interferonopathies and for cancer treatment.

Keywords:  DNA replication dynamics; cGAS-STING; fork processing; fork progression; fork reversal; inflammation

DOI:  https://doi.org/10.3389/fcell.2021.702584
Nat Commun. 2021 Jul 16. 12(1): 4373

Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage.

Xiao Wu, Bin Wang.

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.

DOI: https://doi.org/10.1038/s41467-021-24665-w
Front Cell Dev Biol. 2021 ;9 699966

Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA.

Qiuzhen Li, Kata Dudás, Gabriella Tick, Lajos Haracska.

  DNA interstrand crosslinks (ICLs) are covalently bound DNA lesions, which are commonly induced by chemotherapeutic drugs, such as cisplatin and mitomycin C or endogenous byproducts of metabolic processes. This type of DNA lesion can block ongoing RNA transcription and DNA replication and thus cause genome instability and cancer. Several cellular defense mechanism, such as the Fanconi anemia pathway have developed to ensure accurate repair and DNA replication when ICLs are present. Various structure-specific nucleases and translesion synthesis (TLS) polymerases have come into focus in relation to ICL bypass. Current models propose that a structure-specific nuclease incision is needed to unhook the ICL from the replication fork, followed by the activity of a low-fidelity TLS polymerase enabling replication through the unhooked ICL adduct. This review focuses on how, in parallel with the Fanconi anemia pathway, PCNA interactions and ICL-induced PCNA ubiquitylation regulate the recruitment, substrate specificity, activity, and coordinated action of certain nucleases and TLS polymerases in the execution of stalled replication fork rescue via ICL bypass.

Keywords:  DNA repair; PCNA ubiquitylation; interstrand crosslink; structure-specific nuclease; translesion synthesis polymerases

DOI:  https://doi.org/10.3389/fcell.2021.699966
DNA Repair (Amst). 2021 Jul 07. pii: S1568-7864(21)00127-0. [Epub ahead of print]105 103171

Sp1-dependent recruitment of the histone acetylase p300 to DSBs facilitates chromatin remodeling and recruitment of the NHEJ repair factor Ku70.

Michelle L Swift, Kate Beishline, Jane Azizkhan-Clifford.

  In response to DNA damage, most factors involved in damage recognition and repair are tightly regulated to ensure proper repair pathway choice. Histone acetylation at DNA double strand breaks (DSBs) by p300 histone acetyltransferase (HAT) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that phosphorylation of Sp1 by ATM increases its interaction with p300 and that Sp1-dependent recruitment of p300 to DSBs is necessary to modify the histones associated with p300 activity and NHEJ repair factor recruitment and repair. p300 is known to acetylate multiple residues on histones H3 and H4 necessary for NHEJ. Acetylation of H3K18 by p300 is associated with the recruitment of the SWI/SNF chromatin remodeling complex and Ku70 to DSBs for NHEJ repair. Depletion of Sp1 results in decreased acetylation of lysines on histones H3 and H4. Specifically, cells depleted of Sp1 display defects in the acetylation of H3K18, resulting in defective SWI/SNF and Ku70 recruitment to DSBs. These results shed light on mechanisms by which chromatin remodelers are regulated to ensure activation of the appropriate DSB repair pathway.

Keywords:  DNA damage; Double strand break repair; NHEJ; Sp1; p300

DOI:  https://doi.org/10.1016/j.dnarep.2021.103171
Cell Rep. 2021 Jul 13. pii: S2211-1247(21)00781-6. [Epub ahead of print]36(2): 109383

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.

Shin-Ya Isobe, Shin-Ichiro Hiraga, Koji Nagao, Hiroyuki Sasanuma, Anne D Donaldson, Chikashi Obuse.

  DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.

Keywords:  CtIP; HR; MRN; NHEJ; Olaparib; PP1; Pathway choice; RIF1; Resection; Shieldin

DOI:  https://doi.org/10.1016/j.celrep.2021.109383
Curr Opin Genet Dev. 2021 Jul 13. pii: S0959-437X(21)00079-4. [Epub ahead of print]71 27-33

DNA helicases in homologous recombination repair.

Dana Branzei, Barnabas Szakal.

Helicases are in the spotlight of DNA metabolism and are critical for DNA repair in all domains of life. At their biochemical core, they bind and hydrolyze ATP, converting this energy to translocate unidirectionally, with different strand polarities and substrate binding specificities, along one strand of a nucleic acid. In doing so, DNA and RNA helicases separate duplex strands or remove nucleoprotein complexes, affecting DNA repair and the architecture of replication forks. In this review, we focus on recent advances on the roles and regulations of DNA helicases in homologous recombination repair, a critical pathway for mending damaged chromosomes and for ensuring genome integrity.

DOI: https://doi.org/10.1016/j.gde.2021.06.009
Oncogenesis. 2021 Jul 13. 10(7): 49

CtBP1/2 differentially regulate genomic stability and DNA repair pathway in high-grade serous ovarian cancer cell.

YingYing He, Zhicheng He, Jian Lin, Cheng Chen, Yuanzhi Chen, Shubai Liu.

The C-terminal binding proteins (CtBPs), CtBP1 and CtBP2, are transcriptional co-repressor that interacts with multiple transcriptional factors to modulate the stability of chromatin. CtBP proteins were identified with overexpression in the high-grade serous ovarian carcinoma (HGSOC). However, little is known about CtBP proteins' regulatory roles in genomic stability and DNA repair in HGSOC. In this study, we combined whole-transcriptome analysis with multiple research methods to investigate the role of CtBP1/2 in genomic stability. Several key functional pathways were significantly enriched through whole transcription profile analysis of CtBP1/2 knockdown SKOV3 cells, including DNA damage repair, apoptosis, and cell cycle. CtBP1/2 knockdown induced cancer cell apoptosis, increased genetic instability, and enhanced the sensitivity to DNA damage agents, such as γ-irradiation and chemotherapy drug (Carboplatin and etoposide). The results of DNA fiber assay revealed that CtBP1/2 contribute differentially to the integrity of DNA replication track and stability of DNA replication recovery. CtBP1 protects the integrity of stalled forks under metabolic stress condition during prolonged periods of replication, whereas CtBP2 acts a dominant role in stability of DNA replication recovery. Furthermore, CtBP1/2 knockdown shifted the DSBs repair pathway from homologous recombination (HR) to non-homologous end joining (NHEJ) and activated DNA-PK in SKOV3 cells. Interesting, blast through TCGA tumor cases, patients with CtBP2 genetic alternation had a significantly longer overall survival time than unaltered patients. Together, these results revealed that CtBP1/2 play a different regulatory role in genomic stability and DSBs repair pathway bias in serous ovarian cancer cells. It is possible to generate novel potential targeted therapy strategy and translational application for serous ovarian carcinoma patients with a predictable better clinical outcome.

DOI: https://doi.org/10.1038/s41389-021-00344-9
Elife. 2021 Jul 13. pii: e67926. [Epub ahead of print]10

Role of BRCA2 DNA-binding and C-terminal domain on its mobility and conformation in DNA repair.

Maarten W Paul, Arshdeep Sidhu, Yongxin Liang, Sarah E van Rossum-Fikkert, Hanny Odijk, Alex N Zelensky, Roland Kanaar, Claire Wyman.

  BRCA2 is an essential protein in genome maintenance, homologous recombination and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse ES cells and defined their contribution in HR function and dynamic localization in the nucleus, by single particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 was determined by scanning force microscopy. BRCA2 mobility and DNA damage-induced increase in the immobile fraction was largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced homologous recombination function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.

Keywords:  biochemistry; cell biology; chemical biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.67926
Nat Commun. 2021 07 12. 12(1): 4255

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.

Rohit Prakash, Thomas Sandoval, Florian Morati, Jennifer A Zagelbaum, Pei-Xin Lim, Travis White, Brett Taylor, Raymond Wang, Emilie C B Desclos, Meghan R Sullivan, Hayley L Rein, Kara A Bernstein, Przemek M Krawczyk, Jean Gautier, Mauro Modesti, Fabio Vanoli, Maria Jasin.

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

DOI: https://doi.org/10.1038/s41467-021-24205-6
DNA Repair (Amst). 2021 Jun 30. pii: S1568-7864(21)00126-9. [Epub ahead of print]105 103170

The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair.

Carel Fijen, Eli Rothenberg.

  Formation of biomolecular condensates is increasingly recognized as a mechanism employed by cells to deal with stress and to optimize enzymatic reactions. Recent studies have characterized several DNA repair foci as phase-separated condensates, behaving like liquid droplets. Concomitantly, the apparent importance of long non-coding RNAs and RNA-binding proteins for the repair of double-strand breaks has raised many questions about their exact contribution to the repair process. Here we discuss how RNA molecules can participate in condensate formation and how RNA-binding proteins can act as molecular scaffolds. We furthermore summarize our current knowledge about how properties of condensates can influence the choice of repair pathway (homologous recombination or non-homologous end joining) and identify the open questions in this field of emerging importance.

Keywords:  Biomolecular condensates; DNA damage foci; Double-strand break repair; LLPS; lncRNA

DOI:  https://doi.org/10.1016/j.dnarep.2021.103170
Mutat Res. 2021 Jul;pii: S1383-5718(21)00063-2. [Epub ahead of print]867 503372

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

Tamara Murmann-Konda, Aashish Soni, Martin Stuschke, George Iliakis.

  We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Keywords:  Classical non-homologous end joining (c-NHEJ); DNA Double Strand Breaks (DSB); DSB repair pathway choice; G(2) chromatid breaks (CHROMATID BREAKS); Homologous recombination (HR); Ionizing Radiation (IR); Premature chromosome condensation (PCC)

DOI:  https://doi.org/10.1016/j.mrgentox.2021.503372
Cancer Treat Rev. 2021 Jul 07. pii: S0305-7372(21)00104-3. [Epub ahead of print]99 102256

Repositioning PARP inhibitors in the treatment of thoracic malignancies.

Francesco Passiglia, Maria Lucia Reale, Valeria Cetoretta, Elena Parlagreco, Francesca Jacobs, Angela Listì, Luisella Righi, Paolo Bironzo, Silvia Novello, Giorgio Vittorio Scagliotti.

  The evaluation of the homologous recombination repair (HRR) status is emerging as a predictive tumor agnostic biomarker for poly (ADP-ribose) polymerase (PARP) inhibition across different tumor types and testing for HRR-signature is currently a developing area with promising therapeutic implications. Treatment with PARP inhibitors (PARPi) either as single agent or in combination with chemotherapy have shown so far limited activity in patients with thoracic malignancies. A deeper understanding of the biological background underlying HRR-deficient tumors, along with the recent advent of new effective targeted and immunotherapeutic agents, prompted the design of a new generation of clinical trials investigating novel PARPi-combinations in patients with lung cancer as well as malignant pleural mesothelioma. In this review we briefly summarize the biological basis of the DNA damage response pathway inhibition and provide an updated and detailed overview of clinical trials testing different PARPi-combinations strategies in patients with thoracic malignancies.

Keywords:  Homologous recombination repair; Malignant pleural mesothelioma; Non-small cell lung cancer; PARP; Small cell lung cancer

DOI:  https://doi.org/10.1016/j.ctrv.2021.102256
Oncogene. 2021 Jul 14.

CRIP1 cooperates with BRCA2 to drive the nuclear enrichment of RAD51 and to facilitate homologous repair upon DNA damage induced by chemotherapy.

Huiying Sun, Rui Zhou, Yannan Zheng, Zhaowei Wen, Dingling Zhang, Dongqiang Zeng, Jianhua Wu, Zhenhua Huang, Xiaoxiang Rong, Na Huang, Li Sun, Jianping Bin, Yulin Liao, Min Shi, Wangjun Liao.

Homologous recombination (HR) repair is an important determinant of chemosensitivity. However, the mechanisms underlying HR regulation remain largely unknown. Cysteine-rich intestinal protein 1 (CRIP1) is a member of the LIM/double-zinc finger protein family and is overexpressed and associated with prognosis in several tumor types. However, to date, the functional role of CRIP1 in cancer biology is poorly understood. Here we found that CRIP1 downregulation causes HR repair deficiency with concomitant increase in cell sensitivity to cisplatin, epirubicin, and the poly ADP-ribose polymerase (PARP) inhibitor olaparib in gastric cancer cells. Mechanistically, upon DNA damage, CRIP1 is deubiquitinated and upregulated by activated AKT signaling. CRIP1, in turn, promotes nuclear enrichment of RAD51, which is a prerequisite step for HR commencement, by stabilizing BRCA2 to counteract FBXO5-targeted RAD51 degradation and by binding to the core domain of RAD51 (RAD51184-257) in coordination with BRCA2, to facilitate nuclear export signal masking interactions between BRCA2 and RAD51. Moreover, through mass spectrometry screening, we found that KPNA4 is at least one of the carriers controlling the nucleo-cytoplasmic distribution of the CRIP1-BRCA2-RAD51 complex in response to chemotherapy. Consistent with these findings, RAD51 inhibitors block the CRIP1-mediated HR process, thereby restoring chemotherapy sensitivity of gastric cancer cells with high CRIP1 expression. Analysis of patient specimens revealed an abnormally high level of CRIP1 expression in GC tissues compared to that in the adjacent normal mucosa and a significant negative association between CRIP1 expression and survival time in patient cohorts with different types of solid tumors undergoing genotoxic treatments. In conclusion, our study suggests an essential function of CRIP1 in promoting HR repair and facilitating gastric cancer cell adaptation to genotoxic therapy.

DOI: https://doi.org/10.1038/s41388-021-01932-0
Am J Cancer Res. 2021 ;11(6): 2838-2852

NONO phase separation enhances DNA damage repair by accelerating nuclear EGFR-induced DNA-PK activation.

Xin-Juan Fan, Yun-Long Wang, Wan-Wen Zhao, Shao-Mei Bai, Yan Ma, Xin-Ke Yin, Li-Li Feng, Wei-Xing Feng, Ying-Nai Wang, Quentin Liu, Mien-Chie Hung, Xiang-Bo Wan.

Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy.

Keywords: DNA repair; NONO; Phase separation; nuclear EGFR; radioresistance
Mol Cell Biol. 2021 Jul 12. MCB0008121

MEKK1-dependent activation of the CRL4 complex is important for DNA damage-induced degradation of p21 and DDB2 and cell survival.

Susanne Bacher, Hilda Stekman, Carla M Farah, Annika Karger, Michael Kracht, M Lienhard Schmitz.

Cullin-4 ubiquitin ligase (CRL4) complexes are differentially composed and highly dynamic protein assemblies that control many biological processes including the global genome nucleotide excision repair (GG-NER) pathway. Here we identified the kinase mitogen-activated protein kinase kinase kinase 1 (MEKK1) as a novel constitutive interactor of a cytosolic CRL4 complex that disassembles after DNA damage due to the Caspase-mediated cleavage of MEKK1. The kinase activity of MEKK1 was important to trigger auto-ubiquitination of the CRL4 complex by K48- and K63-linked ubiquitin chains. MEKK1 knockdown prohibited DNA damage-induced degradation of the CRL4 component DNA-damage binding protein 2 (DDB2) and the CRL4 substrate p21 and also cell recovery and survival. A ubiquitin replacement strategy revealed a contribution of K63-branched ubiquitin chains for DNA damage-induced DDB2/p21 decay, cell cycle regulation and cell survival. These data might have also implications for cancer, as frequently occurring mutations of MEKK1 might have an impact on genome stability and the therapeutic efficacy of CRL4-dependent immunomodulatory drugs such as thalidomide-derivatives.

DOI: https://doi.org/10.1128/MCB.00081-21
Mol Cell. 2021 Jul 15. pii: S1097-2765(21)00502-5. [Epub ahead of print]81(14): 2873-2874

Stochastic initiation of DNA replication across the human genome.

David M MacAlpine.

Wang et al. (2021) comprehensively map DNA replication initiation events across the human genome using single-molecule optical resolution mapping and find that initiation events are randomly distributed across broad initiation zones that are only utilized in a stochastic fashion across a population of cells.

DOI: https://doi.org/10.1016/j.molcel.2021.06.022
Sci Rep. 2021 Jul 15. 11(1): 14528

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Haibin Qian, Ron A Hoebe, Michel R Faas, Marc Jan van Goethem, Emiel R van der Graaf, Christoph Meyer, Harry Kiewiet, Sytze Brandenburg, Przemek M Krawczyk.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

DOI: https://doi.org/10.1038/s41598-021-92950-1
Proc Natl Acad Sci U S A. 2021 Jul 13. pii: e2107483118. [Epub ahead of print]118(28):

The intersection of DNA replication with antisense 3' RNA processing in Arabidopsis FLC chromatin silencing.

Colette L Baxter, Saša Šviković, Julian E Sale, Caroline Dean, Silvia Costa.

  How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.

Keywords:  Arabidopsis; DNA replication; chicken DT40; chromatin silencing; transcription

DOI:  https://doi.org/10.1073/pnas.2107483118
EMBO J. 2021 Jul 16. e108053

TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans.

Yisui Xia, Ryo Fujisawa, Tom D Deegan, Remi Sonneville, Karim P M Labib.

  The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS-TIPIN, CUL-2LRR-1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM-7 subunit of CMG. Subsequently, the UBXN-3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4. We show that UBXN-3 is important in vivo for replisome disassembly in the absence of TIMELESS-TIPIN. Correspondingly, co-depletion of UBXN-3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN-3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.

Keywords:  CDC-48; CMG helicase; CUL-2LRR-1; DNA replication termination; TIMELESS-TIPIN; UBXN-3

DOI:  https://doi.org/10.15252/embj.2021108053
Sci Adv. 2021 Jul;pii: eabg0076. [Epub ahead of print]7(29):

The mechanism of gap creation by a multifunctional nuclease during base excision repair.

Jungmin Yoo, Donghun Lee, Hyeryeon Im, Sangmi Ji, Sanghoon Oh, Minsang Shin, Daeho Park, Gwangrog Lee.

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3' end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.

DOI: https://doi.org/10.1126/sciadv.abg0076
Nat Commun. 2021 07 09. 12(1): 4227

mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis.

Rui Liu, Lin-Wen Zeng, Rong Gong, Fanen Yuan, Hong-Bing Shu, Shu Li.

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.

DOI: https://doi.org/10.1038/s41467-021-24321-3
Am J Cancer Res. 2021 ;11(6): 2975-2989

Pterostilbene inhibits hepatocellular carcinoma proliferation and HBV replication by targeting ribonucleotide reductase M2 protein.

Rui Wang, Zhijian Xu, Jiaping Tian, Qian Liu, Jingwen Dong, Lijuan Guo, Boning Hai, Xia Liu, Hangping Yao, Zhi Chen, Junjie Xu, Lijun Zhu, Haiyi Chen, Tingjun Hou, Weiliang Zhu, Jimin Shao.

Hepatocellular carcinoma (HCC), one of the most deadly diseases all around the world. HBV infection is a causative factor of HCC and closely associated with HCC development. Ribonucleotide reductase (RR) is a key enzyme for cellular DNA synthesis and RR small subunit M2 (RRM2) is highly upregulated in HCC with poor survival rates. We have previously shown that HBV can activate the expression of RRM2 and the activity of RR enzyme for the viral DNA replication in host liver cells. Thus, RRM2 may be an important therapeutic target for HCC and HBV-related HCC. Pterostilbene, a natural plant component, potently inhibited in vitro RR enzyme activity with the IC50 of about 0.62 μM through interacting with RRM2 protein, which was much higher than current RRM2 inhibitory drugs. Pterostilbine inhibited cell proliferation with an MTT IC50 of about 20-40 μM in various HCC cell lines, causing DNA synthesis inhibition, cell cycle arrest at S phase, and accordingly apoptosis. On the other hand, the compound significantly inhibited HBV DNA replication in HBV genome integrated and newly transfected HCC cells, and the EC50 for inhibiting HBV replication was significantly lower than the IC50 for inhibiting HCC proliferation. Notably, pterostilbene possessed a similar inhibitory activity in sorafenib and lamivudine resistant HCC cells. Moreover, the inhibitory effects of pterostilbine against HCC proliferation and HBV replication were significantly reversed by addition of dNTP precursors, suggesting that RR was the intracellular target of the compound. Finally, pterostilbine effectively inhibited HCC xenograft growth with a relatively low toxicity in nude mouse experiments. This study demonstrates that pterostilbene is a novel potent RR inhibitor by targeting RRM2. It can simultaneously inhibit HCC proliferation and HBV replication with a potential new use for treatment of HCC and HBV-related HCC.

Keywords: DNA synthesis inhibition; Hepatocellular carcinoma (HCC); hepatitis B virus related hepatocellular carcinoma (HBV-related HCC); pterostilbene; ribonucleotide reductase small subunit M2 (RRM2)
Dev Cell. 2021 Jul 09. pii: S1534-5807(21)00517-7. [Epub ahead of print]

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Richa B Shah, Jennifer L Kernan, Anya van Hoogstraten, Kiyohiro Ando, Yuanyuan Li, Alicia L Belcher, Ivy Mininger, Andrei M Bussenault, Renuka Raman, Ramanagouda Ramanagoudr-Bhojappa, Tony T Huang, Alan D D'Andrea, Settara C Chandrasekharappa, Aneel K Aggarwal, Ruth Thompson, Samuel Sidi.

  Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Keywords:  DNA interstrand crosslink; DNA repair; FANCD2; FANCI; PIDD1; PIDDosome; apoptosis; molecular switch; monoubiquitination; repair failure

DOI:  https://doi.org/10.1016/j.devcel.2021.06.010