bims-numges 2022-05-22 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–05–22
thirty-two papers selected by
Sean Rudd, Karolinska Institutet

Targeting the DNA damage response and repair in cancer through nucleotide metabolism.
Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.
Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.
Exploiting replication gaps for cancer therapy.
Fast and efficient DNA replication with purified human proteins.
The BRCA2 and CDKN1A-interacting protein (BCCIP) stabilizes stalled replication forks and prevents degradation of nascent DNA.
DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells.
DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks.
DNA damage response drugs for cancer yield continued synthetic lethality learnings.
DNA damage and repair in the hematopoietic system.
PWWP2B promotes DNA end resection and homologous recombination.
New twists to the ALTernative endings at telomeres.
Tobacco-specific nitrosamine 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) causes DNA damage and impaired replication/transcription in human lung cells.
DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.
A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress.
A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases.
Fanconi anemia: current insights regarding epidemiology, cancer, and DNA repair.
Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor.
Targeting of PI3K/AKT signaling and DNA damage response in acute myeloid leukemia: a novel therapeutic strategy to boost chemotherapy response and overcome resistance.
LC-MS/MS for Assessing the Incorporation and Repair of N2-Alkyl-2'-deoxyguanosine in Genomic DNA.
The Bidirectional Link Between RNA Cleavage and Polyadenylation and Genome Stability: Recent Insights From a Systematic Screen.
XPG in the Nucleotide Excision Repair and Beyond: a study on the different functional aspects of XPG and its associated diseases.
Fueling genome maintenance: On the versatile roles of NAD+ in preserving DNA integrity.
DNA fiber combing protocol using in-house reagents and coverslips to analyze replication fork dynamics in mammalian cells.
Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.
Targeting DNA damage response in cardiovascular diseases: from pathophysiology to therapeutic implications.
Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery.
Translesion synthesis of apurinic/apyrimidic site analogues by Y-family DNA polymerase Dbh from.
Characterization of uridine-cytidine kinase like-1 nucleoside kinase activity and its role in tumor growth.
Discovery and identification of genes involved in DNA damage repair in yeast.
Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis.
Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis.

Mol Oncol. 2022 May 18.

Targeting the DNA damage response and repair in cancer through nucleotide metabolism.

Thomas Helleday, Sean G Rudd.

  Exploitation of the DNA damage response and DNA repair proficiency of cancer cells is an important anti-cancer strategy. The replication and repair of DNA is dependent upon the supply of deoxynucleoside triphosphate (dNTP) building blocks, which are produced and maintained by nucleotide metabolic pathways. Enzymes within these pathways can be promising targets to selectively induce toxic DNA lesions in cancer cells. These same pathways also activate antimetabolites, an important group of chemotherapies that disrupt both nucleotide and DNA metabolism to induce DNA damage in cancer cells. Thus, dNTP metabolic enzymes can also be targeted to refine the use of these chemotherapeutics, many of which remain standard-of-care in common cancers. In this review article, we will discuss both these approaches exemplified by the enzymes MTH1, MTHFD2, and SAMHD1.

Keywords:  Cancer; DNA damage response; MTH1; MTHFD2; SAMHD1; dNTP metabolism

DOI:  https://doi.org/10.1002/1878-0261.13227
Nat Commun. 2022 May 16. 13(1): 2698

Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.

Mona Hoseini Soflaee, Rushendhiran Kesavan, Umakant Sahu, Alpaslan Tasdogan, Elodie Villa, Zied Djabari, Feng Cai, Diem H Tran, Hieu S Vu, Eunus S Ali, Halie Rion, Brendan P O'Hara, Sherwin Kelekar, James Hughes Hallett, Misty Martin, Thomas P Mathews, Peng Gao, John M Asara, Brendan D Manning, Issam Ben-Sahra, Gerta Hoxhaj.

Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.

DOI: https://doi.org/10.1038/s41467-022-30362-z
EMBO J. 2022 May 16. e110632

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Sabrina X Van Ravenstein, Kavi P Mehta, Tamar Kavlashvili, Jo Ann W Byl, Runxiang Zhao, Neil Osheroff, David Cortez, James M Dewar.

  Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

Keywords:  DNA Damage; DNA replication; cancer; chemotherapy; topoisomerase

DOI:  https://doi.org/10.15252/embj.2022110632
Mol Cell. 2022 May 04. pii: S1097-2765(22)00386-0. [Epub ahead of print]

Exploiting replication gaps for cancer therapy.

Ke Cong, Sharon B Cantor.

  Defects in DNA double-strand break repair are thought to render BRCA1 or BRCA2 (BRCA) mutant tumors selectively sensitive to PARP inhibitors (PARPis). Challenging this framework, BRCA and PARP1 share functions in DNA synthesis on the lagging strand. Thus, BRCA deficiency or "BRCAness" could reflect an inherent lagging strand problem that is vulnerable to drugs such as PARPi that also target the lagging strand, a combination that generates a toxic accumulation of replication gaps.

Keywords:  BRCA cancer; PARP inhibitor; cancer therapy; chemoresistance; single-stranded DNA; synthetic lethality

DOI:  https://doi.org/10.1016/j.molcel.2022.04.023
Nature. 2022 May 18.

Fast and efficient DNA replication with purified human proteins.

Yasemin Baris, Martin R G Taylor, Valentina Aria, Joseph T P Yeeles.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.

DOI: https://doi.org/10.1038/s41586-022-04759-1
FEBS Lett. 2022 May 19.

The BRCA2 and CDKN1A-interacting protein (BCCIP) stabilizes stalled replication forks and prevents degradation of nascent DNA.

Bhawna Singh, Shalini Roy Chowdhury, Mohammad Shoaib Mansuri, Saraswathi Jayarajan Pillai, Sonam Mehrotra.

  DNA replication stress is characterized by impaired replication fork progression, causing some of the replication forks to collapse and form DNA breaks. It is a primary cause of genomic instability leading to oncogenic transformation. The repair-independent functions of the proteins RAD51 and BRCA2, which are involved in homologous recombination (HR)-mediated DNA repair, are crucial for protecting nascent DNA strands from nuclease-mediated degradation. The BRCA2 and CDKN1A-interacting protein (BCCIP) associates with BRCA2 and RAD51 during HR-mediated DNA repair. Here, we investigated the role of BCCIP during the replication stress response. We find that in the presence of replication stress, BCCIP deficiency increases replication fork stalling and results in DNA double-strand break formation. We show that BCCIP is recruited to stalled replication forks and prevents MRE11 nuclease-mediated degradation of nascent DNA strands.

Keywords:  BCCIP; Replication stress; genome stability; replication fork

DOI:  https://doi.org/10.1002/1873-3468.14406
Elife. 2022 May 16. pii: e74700. [Epub ahead of print]11

DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells.

Faith C Fowler, Bo-Ruei Chen, Nicholas Zolnerowich, Wei Wu, Raphael Pavani, Jacob Paiano, Chelsea Peart, Zulong Chen, André Nussenzweig, Barry P Sleckman, Jessica K Tyler.

  DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.

Keywords:  DNA double strand breaks; DNA end resection; DNA-PK; RPA; chromosomes; gene expression; genome stability; mammalian cells; mouse

DOI:  https://doi.org/10.7554/eLife.74700
Nucleic Acids Res. 2022 May 17. pii: gkac375. [Epub ahead of print]

DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks.

Huiming Lu, Junhong Guan, Shih-Ya Wang, Guo-Min Li, Vilhelm A Bohr, Anthony J Davis.

Non-homologous end joining (NHEJ) is the major pathway that mediates the repair of DNA double-strand breaks (DSBs) generated by ionizing radiation (IR). Previously, the DNA helicase RECQL4 was implicated in promoting NHEJ, but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PKcs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PKcs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PKcs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PKcs is required for accumulation of RECQL4 to DSBs and that DNA-PKcs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PKcs in NHEJ.

DOI: https://doi.org/10.1093/nar/gkac375
Nat Rev Drug Discov. 2022 May 17.

DNA damage response drugs for cancer yield continued synthetic lethality learnings.

Asher Mullard.

DOI: https://doi.org/10.1038/d41573-022-00092-4
Acta Biochim Biophys Sin (Shanghai). 2022 Jan 25.

DNA damage and repair in the hematopoietic system.

Niu Li, Hongzhu Chen, Jian Wang.

  Although hematopoietic stem cells (HSCs) in the bone marrow are in a state of quiescence, they harbor the self-renewal capacity and the pluripotency to differentiate into mature blood cells when needed, which is key to maintain hematopoietic homeostasis. Importantly, HSCs are characterized by their long lifespan (.., up to for mice), display characteristics of aging, and are vulnerable to various endogenous and exogenous genotoxic stresses. Generally, DNA damage in HSCs is endogenous, which is typically induced by reactive oxygen species (ROS), aldehydes, and replication stress. Mammalian cells have evolved a complex and efficient DNA repair system to cope with various DNA lesions to maintain genomic stability. The repair machinery for DNA damage in HSCs has its own characteristics. For instance, the Fanconi anemia (FA)/BRCA pathway is particularly important for the hematopoietic system, as it can limit the damage caused by DNA inter-strand crosslinks, oxidative stress, and replication stress to HSCs to prevent FA occurrence. In addition, HSCs prefer to utilize the classical non-homologous end-joining pathway, which is essential for the V(D)J rearrangement in developing lymphocytes and is involved in double-strand break repair to maintain genomic stability in the long-term quiescent state. In contrast, the base excision repair pathway is less involved in the hematopoietic system. In this review, we summarize the impact of various types of DNA damage on HSC function and review our knowledge of the corresponding repair mechanisms and related human genetic diseases.

Keywords:  DNA inter-strand crosslink; FA/BRCA pathway; c-NHEJ; hematopoietic stem cell; oxidative damage; replication stress

DOI:  https://doi.org/10.3724/abbs.2022053
EMBO Rep. 2022 May 18. e53492

PWWP2B promotes DNA end resection and homologous recombination.

Min Kyung Ju, Joo Rak Lee, Yeonsong Choi, Seon Young Park, Hee Jung Sul, Hee Jin Chung, Soyeong An, Semin Lee, Eunyoung Jung, Bohyun Kim, Bo Youn Choi, Bum Jun Kim, Hyeong Su Kim, Hyun Lim, Ho Suk Kang, Jae Seung Soh, Kyungjae Myung, Kab Choong Kim, Ji Woong Cho, Jinwon Seo, Tae Moon Kim, Ja Yil Lee, Yonghwan Kim, Hongtae Kim, Dae Young Zang.

  Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.

Keywords:  PWWP2B; UHRF1; end resection; gastric cancer; homologous recombination

DOI:  https://doi.org/10.15252/embr.202153492
DNA Repair (Amst). 2022 May 13. pii: S1568-7864(22)00075-1. [Epub ahead of print]115 103342

New twists to the ALTernative endings at telomeres.

Ragini Bhargava, Michelle Lee Lynskey, Roderick J O'Sullivan.

  Activation of a telomere maintenance mechanism is key to achieving replicative immortality. Alternative Lengthening of Telomeres (ALT) is a telomerase-independent pathway that hijacks the homologous recombination pathways to elongate telomeres. Commitment to ALT is often associated with several hallmarks including long telomeres of heterogenous lengths, mutations in histone H3.3 or the ATRX/DAXX histone chaperone complex, and incorporation of non-canonical telomere sequences. The consequences of these genetic and epigenetic changes include enhanced replication stress and the presence of transcriptionally permissive chromatin, which can result in replication-associated DNA damage. Here, we detail the molecular mechanisms that are critical to repairing DNA damage at ALT telomeres, including the BLM Helicase, which acts at several steps in the ALT process. Furthermore, we discuss the emerging findings related to the telomere-associated RNA, TERRA, and its roles in maintaining telomeric integrity. Finally, we review new evidence for therapeutic interventions for ALT-positive cancers which are rooted in understanding the molecular underpinnings of this process.

Keywords:  ALT; Cancer; Chromatin; TERRA; Telomere

DOI:  https://doi.org/10.1016/j.dnarep.2022.103342
PLoS One. 2022 ;17(5): e0267839

Tobacco-specific nitrosamine 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) causes DNA damage and impaired replication/transcription in human lung cells.

Altaf H Sarker, Bo Hang.

Thirdhand smoke (THS) is a newly described health hazard composed of toxicants, mutagens and carcinogens, including nicotine-derived tobacco specific nitrosamines (TSNAs), one of which is 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA). Although TSNAs are generally potent carcinogens, the risk of NNA, which is specific to THS, is poorly understood. We recently reported that THS exposure-induced adverse impact on DNA replication and transcription with implications in the development of cancer and other diseases. Here, we investigated the role of NNA in THS exposure-induced harmful effects on fundamental cellular processes. We exposed cultured human lung epithelial BEAS-2B cells to NNA. The formation of DNA base damages was assessed by Long Amplicon QPCR (LA-QPCR); DNA double-strand breaks (DSBs) and NNA effects on replication and transcription by immunofluorescence (IF); and genomic instability by micronuclei (MN) formation. We found increased accumulation of oxidative DNA damage and DSBs as well as activation of DNA damage response pathway, after exposure of cells to NNA. Impaired S phase progression was also evident. Consistent with these results, we found increased MN formation, a marker of genomic instability, in NNA-exposed cells. Furthermore, ongoing RNA synthesis was significantly reduced by NNA exposure, however, RNA synthesis resumed fully after a 24h recovery period only in wild-type cells but not in those deficient in transcription-coupled nucleotide excision repair (TC-NER). Importantly, these cellular effects are common with the THS-exposure induced effects. Our findings suggest that NNA in THS could be a contributing factor for THS exposure-induced adverse health effect.

DOI: https://doi.org/10.1371/journal.pone.0267839
J Cell Sci. 2022 May 18. pii: jcs.259514. [Epub ahead of print]

DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.

Akira Kawamura, Saishu Yoshida, Katsuhiko Aoki, Yuya Shimoyama, Kohji Yamada, Kiyotsugu Yoshida.

  Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), an ubiquitin-like protein, is an essential regulator for the DNA damage response. Numerous studies have shown that neddylation dysfunction causes several human diseases, such as cancer. Hence, clarifying the regulatory mechanism for neddylation could provide insight into the mechanism of genome stability underlying the DNA damage response (DDR) and carcinogenesis. Here, we demonstrate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a novel regulator of neddylation and maintains genome stability. Deletion of DYRK2 leads to persistent DNA double-stand breaks (DSBs) and subsequent genome instability. Mechanistically, DYRK2 promotes neddylation through forming a complex with NAE1, which is a component of NEDD8-activating enzyme E1, and maintaining its protein level by suppressing polyubiquitination. The present study is the first to demonstrate that DYRK2 controls neddylation and is necessary for maintaining genome stability.

Keywords:  DNA damage response; DNA double-stand breaks; DYRK2; Genome stability; NEDD8; Neddylation

DOI:  https://doi.org/10.1242/jcs.259514
Sci Rep. 2022 May 17. 12(1): 8134

A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress.

Sinem Demirbag-Sarikaya, Yunus Akkoc, Sıla Turgut, Secil Erbil-Bilir, Nur Mehpare Kocaturk, Joern Dengjel, Devrim Gozuacik.

The maintenance of cellular homeostasis in living organisms requires a balance between anabolic and catabolic reactions. Macroautophagy (autophagy herein) is determined as one of the major catabolic reactions. Autophagy is an evolutionarily conserved stress response pathway that is activated by various insults including DNA damage. All sorts of damage to DNA potentially cause loss of genetic information and trigger genomic instability. Most of these lesions are repaired by the activation of DNA damage response following DNA repair mechanisms. Here we describe, a novel protein complex containing the autophagy protein ATG5 and the non-homologous end-joining repair system proteins. We discovered for the first time that ATG5 interacted with both Ku80 (XRCC5) and Ku70 (XRCC6). This novel interaction is facilitated mainly via Ku70. Our results suggest that this interaction is dynamic and enhanced upon genotoxic stresses. Strikingly, we identified that ATG5-Ku70 interaction is necessary for DNA repair and effective recovery from genotoxic stress. Therefore, our results are demonstrating a novel, direct, dynamic, and functional interaction between ATG5 and Ku70 proteins that plays a crucial role in DNA repair under genotoxic stress conditions.

DOI: https://doi.org/10.1038/s41598-022-11704-9
Nat Commun. 2022 May 16. 13(1): 2700

A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases.

Inna Rozman Grinberg, Markel Martínez-Carranza, Ornella Bimai, Ghada Nouaïria, Saher Shahid, Daniel Lundin, Derek T Logan, Britt-Marie Sjöberg, Pål Stenmark.

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.

DOI: https://doi.org/10.1038/s41467-022-30328-1
Hum Genet. 2022 May 21.

Fanconi anemia: current insights regarding epidemiology, cancer, and DNA repair.

Jasmine D Peake, Eishi Noguchi.

Fanconi anemia is a genetic disorder that is characterized by bone marrow failure, as well as a predisposition to malignancies including leukemia and squamous cell carcinoma (SCC). At least 22 genes are associated with Fanconi anemia, constituting the Fanconi anemia DNA repair pathway. This pathway coordinates multiple processes and proteins to facilitate the repair of DNA adducts including interstrand crosslinks (ICLs) that are generated by environmental carcinogens, chemotherapeutic crosslinkers, and metabolic products of alcohol. ICLs can interfere with DNA transactions, including replication and transcription. If not properly removed and repaired, ICLs cause DNA breaks and lead to genomic instability, a hallmark of cancer. In this review, we will discuss the genetic and phenotypic characteristics of Fanconi anemia, the epidemiology of the disease, and associated cancer risk. The sources of ICLs and the role of ICL-inducing chemotherapeutic agents will also be discussed. Finally, we will review the detailed mechanisms of ICL repair via the Fanconi anemia DNA repair pathway, highlighting critical regulatory processes. Together, the information in this review will underscore important contributions to Fanconi anemia research in the past two decades.

DOI: https://doi.org/10.1007/s00439-022-02462-9
Bio Protoc. 2022 Mar 05. 12(5): e4337

Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor.

Carolina Dos Santos Passos, Robert E Cohen, Tingting Yao.

  Double-strand breaks (DSBs) are lesions in DNA that, if not properly repaired, can cause genomic instability, oncogenesis, and cell death. Multiple chromatin posttranslational modifications (PTMs) play a role in the DNA damage response to DSBs. Among these, RNF168-mediated ubiquitination of lysines 13 or 15 at the N-terminal tail of histone H2A (H2AK13/15Ub) is essential for the recruitment of effectors of both the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair pathways. Thus, tools and techniques to track the spatiotemporal dynamics of H2AK13/15 ubiquitination at DNA DSBs are important to facilitate studies of DNA repair. Previous work from other groups used the minimal focus-forming region (FFR) of the NHEJ effector 53BP1 to detect H2AK15Ub generated upon damage induced by gamma or laser irradiation in live cells. However, 53BP1-FFR only binds nucleosomes modified with both H2AK15Ub and dimethylation of lysine 20 on histone H4 (H4K20me2); thus, 53BP1-FFR does not recognize H2AK13Ub-nucleosomes or nucleosomes that contain H2AK15Ub but lack methylation of H4K20 (H4K20me0). To overcome this limitation, we developed an avidity-based sensor that binds H2AK13/15Ub without dependence on the methylation status of histone H4K20. This sensor, called Reader1.0, detects DNA damage-associated H2AK13/15Ub in live cells with high sensitivity and selectivity. Here, we present a protocol to detect the formation of H2AK13/15Ub at laser-induced DSBs using Reader1.0 as a live-cell reporter for this histone PTM. Graphic abstract.

Keywords:  DNA damage repair; DSB; H2AK13/15Ub; Laser microirradiation; Live-cell sensor; RNF168-mediated ubiquitination

DOI:  https://doi.org/10.21769/BioProtoc.4337
Cancer Drug Resist. 2021 ;4(4): 984-995

Targeting of PI3K/AKT signaling and DNA damage response in acute myeloid leukemia: a novel therapeutic strategy to boost chemotherapy response and overcome resistance.

Montserrat Estruch, Camilla Vittori, Teresa Muñoz Montesinos, Kristian Reckzeh, Kim Theilgaard-Mönch.

  Resistance of cancer patients to DNA damaging radiation therapy and chemotherapy remains a major problem in the clinic. The current review discusses the molecular mechanisms of therapy resistance in acute myeloid leukemia (AML) conferred by cooperative chemotherapy-induced DNA damage response (DDR) and mutational activation of PI3K/AKT signaling. In addition, strategies to overcome resistance are discussed, with particular focus on studies underpinning the vast potential of therapies combining standard chemotherapy AML regimens with small molecule inhibitors targeting key regulatory hubs at the interface of DDR and oncogenic signaling pathways.

Keywords:  AML; DNA damage response; PI3K/AKT; chemotherapy; resistance

DOI:  https://doi.org/10.20517/cdr.2021.76
Chem Res Toxicol. 2022 May 18.

LC-MS/MS for Assessing the Incorporation and Repair of N2-Alkyl-2'-deoxyguanosine in Genomic DNA.

Su Guo, Lin Li, Kailin Yu, Ying Tan, Yinsheng Wang.

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N2-MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N2-alkyl-dG lesions in mammalian cells.

DOI: https://doi.org/10.1021/acs.chemrestox.2c00101
Front Genet. 2022 ;13 854907

The Bidirectional Link Between RNA Cleavage and Polyadenylation and Genome Stability: Recent Insights From a Systematic Screen.

Stefano Spada, Brian Luke, Sven Danckwardt.

  The integrity of the genome is governed by multiple processes to ensure optimal survival and to prevent the inheritance of deleterious traits. While significant progress has been made to characterize components involved in the DNA Damage Response (DDR), little is known about the interplay between RNA processing and the maintenance of genome stability. Here, we describe the emerging picture of an intricate bidirectional coupling between RNA processing and genome integrity in an integrative manner. By employing insights from a recent large-scale RNAi screening involving the depletion of more than 170 components that direct (alternative) polyadenylation, we provide evidence of bidirectional crosstalk between co-transcriptional RNA 3'end processing and the DDR in a manner that optimizes genomic integrity. We provide instructive examples illustrating the wiring between the two processes and show how perturbations at one end are either compensated by buffering mechanisms at the other end, or even propel the initial insult and thereby become disease-eliciting as evidenced by various disorders.

Keywords:  DNA damage response; aging; alternative polyadenylation; cancer; cleavage and polyadenylation; genome integrity; resillience; systematic screening

DOI:  https://doi.org/10.3389/fgene.2022.854907
Mol Biol Rep. 2022 May 20.

XPG in the Nucleotide Excision Repair and Beyond: a study on the different functional aspects of XPG and its associated diseases.

Riasha Pal, Nilanjan Paul, Deep Bhattacharya, Sudeshna Rakshit, Geetha Shanmugam, Koustav Sarkar.

  Several proteins are involved in DNA repair mechanisms attempting to repair damages to the DNA continuously. One such protein is Xeroderma Pigmentosum Complementation Group G (XPG), a significant component in the Nucleotide Excision Repair (NER) pathway. XPG is accountable for making the 3' incision in the NER, while XPF-ERCC4 joins ERCC1 to form the XPF-ERCC1 complex. This complex makes a 5' incision to eliminate bulky DNA lesions. XPG is also known to function as a cofactor in the Base Excision Repair (BER) pathway by increasing hNth1 activity, apart from its crucial involvement in the NER. Reports suggest that XPG also plays a non-catalytic role in the Homologous Recombination Repair (HRR) pathway by forming higher-order complexes with BRCA1, BRCA2, Rad51, and PALB2, further influencing the activity of these molecules. Studies show that, apart from its vital role in repairing DNA damages, XPG is also responsible for R-loop formation, which facilitates exhibiting phenotypes of Werner Syndrome. Though XPG has a role in several DNA repair pathways and molecular mechanisms, it is primarily a NER protein. Unrepaired and prolonged DNA damage leads to genomic instability and facilitates neurological disorders, aging, pigmentation, and cancer susceptibility. This review explores the vital role of XPG in different DNA repair mechanisms which are continuously involved in repairing these damaged sites and its failure leading to XP-G, XP-G/CS complex phenotypes, and cancer progression.

Keywords:  Base excision repair; Homologous recombination repair, R-loops; Nucleotide excision repair; Xeroderma Pigmentosum complementation group G

DOI:  https://doi.org/10.1007/s11033-022-07324-1
J Biol Chem. 2022 May 17. pii: S0021-9258(22)00477-X. [Epub ahead of print] 102037

Fueling genome maintenance: On the versatile roles of NAD+ in preserving DNA integrity.

Joanna A Ruszkiewicz, Alexander Bürkle, Aswin Mangerich.

  Nicotinamide adenine dinucleotide (NAD+) is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD+ in mechanisms promoting genome maintenance. Numerous NAD+-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD+ serves as a substrate to ADP-ribosyltransferases (ARTs), sirtuins (SIRTs), and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD+ levels.

Keywords:  ARTs; DNA repair; Nicotinamide adenine dinucleotide; PARPs; sirtuins

DOI:  https://doi.org/10.1016/j.jbc.2022.102037
STAR Protoc. 2022 Jun 17. 3(2): 101371

DNA fiber combing protocol using in-house reagents and coverslips to analyze replication fork dynamics in mammalian cells.

Gemma Moore, Judit Jimenez Sainz, Ryan B Jensen.

  DNA fiber combing is a versatile technique that provides insight into replication fork dynamics at single-molecule resolution. DNA fibers are bound to silanized coverslips and combed, which straightens and aligns the fibers along a single axis. Here, we present a DNA fiber combing protocol that does not use commercial kits; we detail the steps to prepare all materials, reagents, and silanized coverslips. We describe the use of DLD-1 cells, but the protocol is amenable to other cell types.

Keywords:  Cell Biology; Molecular Biology; Single-molecule Assays

DOI:  https://doi.org/10.1016/j.xpro.2022.101371
Cancer Res. 2022 May 18. pii: canres.CAN-21-2072-E.2021. [Epub ahead of print]

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

Amrita M Nargund, Chang Xu, Amit Mandoli, Atsushi Okabe, Gao Bin Chen, Kie Kyon Huang, Taotao Sheng, Xiaosai Yao, Jia Ming Nickolas Teo, Raghav Sundar, Yee Jiun Kok, Yi Xiang See, Manjie Xing, Zhimei Li, Chern Han Yong, Aparna Anand, Zul Fazreen A I, Lai Fong Poon, Michelle Shu Wen Ng, Javier Yu Peng Koh, Wen Fong Ooi, Su Ting Tay, Xuewen Ong, Angie Lay Keng Tan, Heike I Grabsch, Melissa Jane Fullwood, Tean Bin Teh, Xuezhi Bi, Atsushi Kaneda, Shang Li, Patrick Tan.

Mutations in the DNA mismatch repair gene MSH2 are causative of microsatellite instability (MSI) in multiple cancers. Here, we discovered that besides its well-established role in DNA repair, MSH2 exerts a novel epigenomic function in gastric cancer (GC). Unbiased CRISPR-based mass spectrometry combined with genome-wide CRISPR functional screening revealed that in early-stage GC MSH2 genomic binding is not randomly distributed but rather is associated specifically with tumor-associated super-enhancers controlling the expression of cell adhesion genes. At these loci, MSH2 genomic binding was required for chromatin rewiring, de novo enhancer-promoter interactions, maintenance of histone acetylation levels, and regulation of cell adhesion pathway expression. The chromatin function of MSH2 was independent of its DNA repair catalytic activity but required MSH6, another DNA repair gene, and recruitment to gene loci by the SWI/SNF chromatin remodeler SMARCA4/BRG1. Loss of MSH2 in advanced GCs was accompanied by deficient cell adhesion pathway expression, epithelial-mesenchymal transition, and enhanced tumorigenesis in vitro and in vivo. However, MSH2-deficient GCs also displayed addiction to BAZ1B, a bromodomain-containing family member, and consequent synthetic lethality to bromodomain and extra-terminal motif (BET) inhibition. Our results reveal a role for MSH2 in GC epigenomic regulation and identify BET inhibition as a potential therapy in MSH2-deficient gastric malignancies.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2072
Cardiovasc Res. 2022 May 16. pii: cvac080. [Epub ahead of print]

Targeting DNA damage response in cardiovascular diseases: from pathophysiology to therapeutic implications.

Lin Wu, James R Sowers, Yingmei Zhang, Jun Ren.

  Cardiovascular diseases (CVDs), arise from a complex interplay among genomic, proteomic, and metabolomic abnormalities. Emerging evidence has recently consolidated the presence of robust DNA damage in a variety of cardiovascular disorders. DNA damage triggers a series of cellular responses termed DNA damage response (DDR) including detection of DNA lesions, cell cycle arrest, DNA repair, cellular senescence and apoptosis, in all organ systems including hearts and vasculature. Although transient DDR in response to temporary DNA damage can be beneficial for cardiovascular function, persistent activation of DDR promotes the onset and development of CVDs. Moreover, therapeutic interventions that target DNA damage and DDR have the potential to attenuate cardiovascular dysfunction and improve disease outcome. In this review, we will discuss molecular mechanisms of DNA damage and repair in the onset and development of CVDs, and explore how DDR in specific cardiac cell types contributes to CVDs. Moreover, we will highlight the latest advances regarding the potential therapeutic strategies targeting DNA damage signaling in CVDs.

Keywords:  Cardiovascular disease; DNA damage; DNA damage response; DNA repair; Therapeutics

DOI:  https://doi.org/10.1093/cvr/cvac080
J Biol Chem. 2022 May 11. pii: S0021-9258(22)00468-9. [Epub ahead of print] 102028

Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery.

Sascha Krakovka, Farahnaz Ranjbarian, Lucas A Luján, Alicia Saura, Nicolai B Larsen, Alejandro Jiménez-González, Anna Reggenti, Hugo D Luján, Staffan G Svärd, Anders Hofer.

Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogues. Here we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes dUMP a dead-end product that is potentially harmful if converted to dUTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against wild-type as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.

DOI: https://doi.org/10.1016/j.jbc.2022.102028
Acta Biochim Biophys Sin (Shanghai). 2022 Apr 25.

Translesion synthesis of apurinic/apyrimidic site analogues by Y-family DNA polymerase Dbh from.

Weiwei Wang, Huan Zhou, Li Peng, Feng Yu, Qin Xu, Qisheng Wang, Jianhua He, Xipeng Liu.

  Apurinic/apyrimidic (AP) sites are severe DNA damages and strongly block DNA extension by major DNA polymerases. Y-family DNA polymerases possess a strong ability to bypass AP sites and continue the DNA synthesis reaction, which is called translesion synthesis (TLS) activity. To investigate the effect of the molecular structure of the AP site on the TLS efficiency of Dbh, a Y-family DNA polymerase from , a series of different AP site analogues (various spacers) are used to characterize the bypass efficiency. We find that not only the molecular structure and atomic composition but also the number and position of AP site analogues determine the TLS efficiency of Dbh. Increasing the spacer length decreases TLS activity. The TLS efficiency also decreases when more than one spacer exists on the DNA template. The position of the AP site analogues is also an important factor for TLS. When the spacer is opposite to the first incorporated dNTPs, the TLS efficiency is the lowest, suggesting that AP sites are largely harmful for the formation of hydrogen bonds. These results deepen our understanding of the TLS activity of Y-family DNA polymerases and provide a biochemical basis for elucidating the TLS mechanism in cells.

Keywords:  AP site analogues; Dbh; Y-family DNA polymerase; s; translesion synthesis

DOI:  https://doi.org/10.3724/abbs.2022045
Biochem J. 2022 May 18. pii: BCJ20210770. [Epub ahead of print]

Characterization of uridine-cytidine kinase like-1 nucleoside kinase activity and its role in tumor growth.

Emily Matchett, Elise C Ambrose, Jacki Kornbluth.

  Uridine-cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein with high sequence similarity to other uridine-cytidine kinases (UCKs). UCKs play an important role in the pyrimidine salvage pathway, catalyzing the phosphorylation of uridine and cytidine to UMP and CMP, respectively. Only two human UCKs have been identified, UCK1 and UCK2. Previous studies have shown both enzymes phosphorylate uridine and cytidine using ATP as the phosphate donor. No studies have evaluated the kinase potential of UCKL-1. We cloned and purified UCKL-1 and found that it successfully phosphorylated uridine and cytidine using ATP as the phosphate donor. The catalytic efficiency (calculated as kcat/KM) was 1.2 x 104 s-1, M-1 for uridine and 0.7 x 104 s-1, M-1 for cytidine. Previously, our lab determined UCKL-1 is upregulated in tumor cells, providing protection against natural killer (NK) cell killing activity. We utilized small interfering RNA (siRNA) to downregulate UCKL-1 in vitro and in vivo to determine the effect of UCKL-1 on tumor growth and metastasis. The downregulation of UCKL-1 in YAC-1 lymphoma cells in vitro resulted in decreased cell counts and increased apoptotic activity. Downregulation of UCKL-1 in K562 leukemia cells in vivo led to decreased primary tumor growth and less tumor cell dissemination and metastasis. These results identify UCKL-1 as a bona fide pyrimidine kinase with the therapeutic potential to be a target for tumor growth inhibition and for diminishing or preventing metastasis.

Keywords:  cancer; metastasis; natural killer; nucleoside kinase; uridine kinase

DOI:  https://doi.org/10.1042/BCJ20210770
Gene. 2022 May 12. pii: S0378-1119(22)00368-7. [Epub ahead of print] 146549

Discovery and identification of genes involved in DNA damage repair in yeast.

Sasi Kumar Jagadeesan, Taylor Potter, Mustafa Al-Gafari, Mohsen Hooshyar, Chamath Minuka Hewapathirana, Sarah Takallou, Maryam Hajikarimlou, Daniel Burnside, Bahram Samanfar, Houman Moteshareie, Myron Smith, Ashkan Golshani.

  DNA repair defects are common in tumour cells and can lead to misrepair of double-strand breaks (DSBs), posing a significant challenge to cellular integrity. The overall mechanisms of DSB have been known for decades. However, the list of the genes that affect the efficiency of DSB repair continues to grow. Additional factors that play a role in DSB repair pathways have yet to be identified. In this study, we present a computational approach to identify novel gene functions that are involved in DNA damage repair in Saccharomyces cerevisiae. Among the primary candidates, GAL7, YMR130W, and YHI9 were selected for further analysis since they had not previously been identified as being active in DNA repair pathways. Originally, GAL7 was linked to galactose metabolism. YHI9 and YMR130W encode proteins of unknown functions. Laboratory testing of deletion strains gal7Δ, ymr130wΔ, and yhi9Δ implicated all 3 genes in Homologous Recombination (HR) and/or Non-Homologous End Joining (NHEJ) repair pathways, and enhanced sensitivity to DNA damage-inducing drugs suggested involvement in the broader DNA damage repair machinery. A subsequent genetic interaction analysis revealed interconnections of these three genes, most strikingly through SIR2, SIR3 and SIR4 that are involved in chromatin regulation and DNA damage repair network.

Keywords:  DNA Damage; DNA Repair; Double-Stranded Breaks; Functional Genomics; Genetic Interaction; Network Biology; Non-homologous end joining; Protein-Protein Interaction; Saccharomyces cerevisiae; Yeast

DOI:  https://doi.org/10.1016/j.gene.2022.146549
Nat Commun. 2022 May 16. 13(1): 2699

Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis.

Nicole Kiweler, Catherine Delbrouck, Vitaly I Pozdeev, Laura Neises, Leticia Soriano-Baguet, Kim Eiden, Feng Xian, Mohaned Benzarti, Lara Haase, Eric Koncina, Maryse Schmoetten, Christian Jaeger, Muhammad Zaeem Noman, Alexei Vazquez, Bassam Janji, Gunnar Dittmar, Dirk Brenner, Elisabeth Letellier, Johannes Meiser.

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

DOI: https://doi.org/10.1038/s41467-022-30363-y
Dev Cell. 2022 May 11. pii: S1534-5807(22)00286-6. [Epub ahead of print]

Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis.

Roxana E Oberkersch, Giovanna Pontarin, Matteo Astone, Marianna Spizzotin, Liaisan Arslanbaeva, Giovanni Tosi, Emiliano Panieri, Sara Ricciardi, Maria Francesca Allega, Alessia Brossa, Paolo Grumati, Benedetta Bussolati, Stefano Biffo, Saverio Tardito, Massimo M Santoro.

  Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation.

Keywords:  angiogenesis; aspartate metabolism; endothelial metabolism; mTOR signalling; tumor angiogenesis

DOI:  https://doi.org/10.1016/j.devcel.2022.04.018