bims-numges 2022-06-12 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒06‒12
35 papers selected by
Sean Rudd
Karolinska Institutet

Watching right and wrong nucleotide insertion captures hidden polymerase fidelity checkpoints.
Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.
Targeting Mechanisms of the DNA Damage Response (DDR) and DNA Repair by Natural Compounds to Improve cAT-Triggered Tumor Cell Death.
Insight into RNA-DNA primer length counting by human primosome.
Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy.
MutL binds to 3' resected DNA ends and blocks DNA polymerase access.
THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1.
DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage.
MCL-1 interacts with MOF and BID to regulate H4K16 acetylation and homologous recombination repair.
Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells.
A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.
Pharmacologic Induction of BRCAness in BRCA-Proficient Cancers: Expanding PARP Inhibitor Use.
Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutants Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes.
The Metabolic and Non-Metabolic Roles of UCK2 in Tumor Progression.
The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.
DNA Damage Response and Repair in Adaptive Immunity.
The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.
TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.
Genome-wide mapping of individual replication fork velocities using nanopore sequencing.
Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process.
Glycolysis addiction compensating for a defective pentose phosphate pathway confers gemcitabine sensitivity in SETD2-deficient pancreatic cancer.
TAS1553, a small molecule subunit interaction inhibitor of ribonucleotide reductase, exhibits antitumor activity by causing DNA replication stress.
Condensates induced by transcription inhibition localize active chromatin to nucleoli.
Telomere-to-telomere human DNA replication timing profiles.
Insights into the Possible Molecular Mechanisms of Resistance to PARP Inhibitors.
Functional RECAP (REpair CAPacity) assay identifies homologous recombination deficiency undetected by DNA-based BRCAness tests.
Concordance of hydrogen peroxide-induced 8-oxo-guanine patterns with two cancer mutation signatures of upper GI tract tumors.
Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks.
Control of a pyrimidine ribonucleotide salvage pathway in Pseudomonas oleovorans.
Cohesin-mediated loop anchors confine the locations of human replication origins.
Core control principles of the eukaryotic cell cycle.
Targeting MUC1-C Suppresses Chronic Activation of Cytosolic Nucleotide Receptors and STING in Triple-Negative Breast Cancer.
Insight into the nucleoside transport and inhibition of human ENT1.
SAMHD1, positively regulated by KLF4, suppresses the proliferation of gastric cancer cells through MAPK p38 signaling pathway.
Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Nat Commun. 2022 Jun 09. 13(1): 3193

Watching right and wrong nucleotide insertion captures hidden polymerase fidelity checkpoints.

Joonas A Jamsen, David D Shock, Samuel H Wilson.

Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability.

DOI: https://doi.org/10.1038/s41467-022-30141-w
Cell Mol Life Sci. 2022 Jun 04. 79(6): 339

Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.

Barbara Guerra, Thomas K Doktor, Sabrina B Frederiksen, Kumar Somyajit, Brage S Andresen.

  The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7-CDC45, ATRIP-ATR-MCM7, and ATR-CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.

Keywords:  CDC45; CK2α; CLSPN; MCM7; S-phase checkpoint

DOI:  https://doi.org/10.1007/s00018-022-04374-3
Molecules. 2022 Jun 01. pii: 3567. [Epub ahead of print]27(11):

Targeting Mechanisms of the DNA Damage Response (DDR) and DNA Repair by Natural Compounds to Improve cAT-Triggered Tumor Cell Death.

Jana Aengenvoort, Marlena Sekeres, Peter Proksch, Gerhard Fritz.

  Recently, we identified secalonic acid F (SA), 5-epi-nakijiquinone Q (NQ) and 5-epi-ilimaquinone (IQ) as natural compounds (NC) affecting mechanisms of the DNA damage response (DDR). Here, we further characterized their effects on DDR, DNA repair and cytotoxicity if used in mono- and co-treatment with conventional anticancer therapeutics (cAT) (cisplatin (Cis), doxorubicin (Doxo)) in vitro. All three NC influence the phosphorylation level of selected DDR-related factors (i.e., pCHK1, pKAP1, pP53, pRPA32) in mono- and/or co-treatment. Both SA and NQ attenuate the Cis- and Doxo-induced G2/M-phase arrest and effectively stimulate caspase-mediated apoptosis. Notably, SA impacts DNA repair as reflected by enhanced steady-state levels of Cis-(1,2-GpG)-DNA adducts and Doxo-induced DNA double-strand breaks (DSB). Moreover, SA decreased the mRNA and protein expression of the homologous recombination (HR)-related DSB repair factors RAD51 and BRCA1. Both SA and NQ promote Cis- and Doxo-induced cytotoxicity in an additive to synergistic manner (CI ≤ 1.0). Summarizing, we conclude that SA promotes cAT-driven caspase-dependent cell death by interfering with DSB repair and DDR-related checkpoint control mechanisms. Hence, SA is considered as the most promising lead compound to evaluate its therapeutic window in forthcoming pre-clinical in vivo studies.

Keywords:  DNA damage response; DNA repair; anticancer drugs; cell death; drug resistance; natural compounds

DOI:  https://doi.org/10.3390/molecules27113567
Nucleic Acids Res. 2022 Jun 11. pii: gkac492. [Epub ahead of print]

Insight into RNA-DNA primer length counting by human primosome.

Andrey G Baranovskiy, Alisa E Lisova, Lucia M Morstadt, Nigar D Babayeva, Tahir H Tahirov.

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA-DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5'-end, and (ii) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

DOI: https://doi.org/10.1093/nar/gkac492
Nucleic Acids Res. 2022 Jun 11. pii: gkac491. [Epub ahead of print]

Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy.

Brian Budke, Alison Zhong, Katherine Sullivan, Chanyoung Park, David I Gittin, Timothy S Kountz, Philip P Connell.

Homologous recombination (HR) serves multiple roles in DNA repair that are essential for maintaining genomic stability, including double-strand DNA break (DSB) repair. The central HR protein, RAD51, is frequently overexpressed in human malignancies, thereby elevating HR proficiency and promoting resistance to DNA-damaging therapies. Here, we find that the non-canonical NF-κB factors p100/52, but not RelB, control the expression of RAD51 in various human cancer subtypes. While p100/p52 depletion inhibits HR function in human tumor cells, it does not significantly influence the proficiency of non-homologous end joining, the other key mechanism of DSB repair. Clonogenic survival assays were performed using a pair DLD-1 cell lines that differ only in their expression of the key HR protein BRCA2. Targeted silencing of p100/p52 sensitizes the HR-competent cells to camptothecin, while sensitization is absent in HR-deficient control cells. These results suggest that p100/p52-dependent signaling specifically controls HR activity in cancer cells. Since non-canonical NF-κB signaling is known to be activated after various forms of genomic crisis, compensatory HR upregulation may represent a natural consequence of DNA damage. We propose that p100/p52-dependent signaling represents a promising oncologic target in combination with DNA-damaging treatments.

DOI: https://doi.org/10.1093/nar/gkac491
Nucleic Acids Res. 2022 Jun 07. pii: gkac432. [Epub ahead of print]

MutL binds to 3' resected DNA ends and blocks DNA polymerase access.

Alessandro Borsellini, Joyce H G Lebbink, Meindert H Lamers.

DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100-1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides is removed from the daughter strand followed by re-synthesis. How these opposite activities are coordinated is poorly understood. Here we show that the Escherichia coli MutL protein binds to the 3' end of the resected strand and blocks access of Pol I and Pol III. The cryo-EM structure of an 85-kDa MutL-DNA complex, determined to 3.7 Å resolution, reveals a unique DNA binding mode that positions MutL at the 3' end of a primer-template, but not at a 5' resected DNA end or a blunt DNA end. Hence, our work reveals a novel role for MutL in the final stages of mismatch repair by preventing premature DNA synthesis during removal of the mismatched strand.

DOI: https://doi.org/10.1093/nar/gkac432
Nucleic Acids Res. 2022 Jun 07. pii: gkac472. [Epub ahead of print]

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1.

Juliette A Kamp, Bennie B L G Lemmens, Ron J Romeijn, Román González-Prieto, Jesper V Olsen, Alfred C O Vertegaal, Robin van Schendel, Marcel Tijsterman.

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.

DOI: https://doi.org/10.1093/nar/gkac472
Cell Death Dis. 2022 Jun 11. 13(6): 546

DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage.

Hsueh-Tzu Shih, Wei-Yi Chen, Hsin-Yen Wang, Tung Chao, Hsien-Da Huang, Chih-Hung Chou, Zee-Fen Chang.

This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.

DOI: https://doi.org/10.1038/s41419-022-04989-1
Cell Biol Int. 2022 Jun 05.

MCL-1 interacts with MOF and BID to regulate H4K16 acetylation and homologous recombination repair.

Abid R Mattoo, John M Jessup.

  The Participation of myeloid cell leukemia-1 (MCL-1), an antiapoptotic protein, in DNA repair and homologous recombination (HR) is not well understood. This study tests whether MCL-1 interacts with Males absent On First (MOF) to regulate H4K16 acetylation that promotes HR repair in response to replication stress induced by Hydroxyurea (HU) treatment. Co-immunoprecipitation of FLAG-MCL-1 from cancer cells treated with HU pulls down a complex of MCL-1, MOF and BH3-interacting domain death agonist (BID). The same complex is pulled down in cells treated with HU that express FLAG-MOF. MCL-1 regulates H4K16 acetylation during HU-induced replication stress since knockdown of MCL-1 decreases H4K16 acetylation while re-expression of MCL-1 restores H4K16 acetylation. Furthermore, knockdown of BID rescues the clonogenic survival in MCL-1 depleted cells in response to replication stress which is associated with decreased Caspase 3/7 activity compared to MCL-1 depleted cells. Cells depleted in both MCL-1 and BID display increased HR repair efficiency by direct repeats-green fluorescent protein assay and in response to HU exhibit increased ATR, Chk1, and RPA phosphorylation relative to MCL-1 depleted cells. This study uncovers that MCL-1 cooperates with MOF and regulates HR repair through H4K16 acetylation. Further, this study determines that MCL-1 and BID cooperate to regulate the crosstalk between HR repair and apoptosis.

Keywords:  H4K16 acetylation; HR repair; MCL-1; MOF; NHEJ

DOI:  https://doi.org/10.1002/cbin.11831
J Biol Chem. 2022 Jun 03. pii: S0021-9258(22)00543-9. [Epub ahead of print] 102102

Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells.

Sida Guo, Jun Fang, Weizhi Xu, Janice Ortega, Chang-Yi Liu, Liya Gu, Zhijie Chang, Guo-Min Li.

  Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair (MMR) has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the MMR processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly co-enriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

Keywords:  SETD2; histone methylation; mismatch repair; oxidative DNA damage

DOI:  https://doi.org/10.1016/j.jbc.2022.102102
Metallomics. 2022 Jun 11. pii: mfac041. [Epub ahead of print]

A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.

Marium Rana, Alessio Perotti, Lucy M Bisset, James D Smith, Emma Lamden, Zahra Khan, Media K Ismail, Katherine Ellis, Katie A Armstrong, Samantha L Hodder, Cosetta Bertoli, Leticia Meneguello, Robertus A M de Bruin, Joanna R Morris, Isolda Romero-Canelon, James H R Tucker, Nikolas J Hodges.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains refractory to existing treatments including the nucleoside analogue gemcitabine. In the current study we demonstrate that an organometallic nucleoside analogue, the ferronucleoside 1-(S, Rp), is cytotoxic in a panel of PDAC cell lines including gemcitabine resistant MIAPaCa2, with IC50 values comparable to cisplatin. Biochemical studies show that the mechanism of action is inhibition of DNA-replication, S-phase cell cycle arrest and stalling of DNA-replication forks which were directly observed at single molecule resolution by DNA-fibre fluorography. In agreement with this, transcriptional changes following treatment with 1-(S, Rp) include activation of three of the four genes (HUS1, RAD1, RAD17) of the 9-1-1 check point complex clamp and two of the three genes (MRE11, NBN) that form the MRN complex as well as activation of multiple downstream targets. Furthermore, there was evidence of phosphorylation of checkpoint kinases 1 and 2 as well as RPA1 and gamma H2AX, all of which are considered biochemical markers of replication stress. Studies in p53 deficient cell lines showed activation of CDKN1A (p21) and GADD45A by 1-(S, Rp) was at least partially independent of p53. In conclusion, because of its potency and activity in gemcitabine resistant cells, 1-(S, Rp) is a promising candidate molecule for development of new treatments for PDAC.

DOI: https://doi.org/10.1093/mtomcs/mfac041
Cancers (Basel). 2022 May 26. pii: 2640. [Epub ahead of print]14(11):

Pharmacologic Induction of BRCAness in BRCA-Proficient Cancers: Expanding PARP Inhibitor Use.

Rachel Abbotts, Anna J Dellomo, Feyruz V Rassool.

  The poly(ADP-ribose) polymerase (PARP) family of proteins has been implicated in numerous cellular processes, including DNA repair, translation, transcription, telomere maintenance, and chromatin remodeling. Best characterized is PARP1, which plays a central role in the repair of single strand DNA damage, thus prompting the development of small molecule PARP inhibitors (PARPi) with the intent of potentiating the genotoxic effects of DNA damaging agents such as chemo- and radiotherapy. However, preclinical studies rapidly uncovered tumor-specific cytotoxicity of PARPi in a subset of cancers carrying mutations in the BReast CAncer 1 and 2 genes (BRCA1/2), which are defective in the homologous recombination (HR) DNA repair pathway, and several PARPi are now FDA-approved for single agent treatment in BRCA-mutated tumors. This phenomenon, termed synthetic lethality, has now been demonstrated in tumors harboring a number of repair gene mutations that produce a BRCA-like impairment of HR (also known as a 'BRCAness' phenotype). However, BRCA mutations or BRCAness is present in only a small subset of cancers, limiting PARPi therapeutic utility. Fortunately, it is now increasingly recognized that many small molecule agents, targeting a variety of molecular pathways, can induce therapeutic BRCAness as a downstream effect of activity. This review will discuss the potential for targeting a broad range of molecular pathways to therapeutically induce BRCAness and PARPi synthetic lethality.

Keywords:  BRCA mutations; BRCAness; DNA repair; PARP inhibitor; cell cycle inhibitor; epigenetic therapy; homologous recombination; kinase inhibitor; synthetic lethality

DOI:  https://doi.org/10.3390/cancers14112640
Genetics. 2022 Jun 10. pii: iyac092. [Epub ahead of print]

Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutants Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes.

Natalie A Lamb, Jonathan Bard, Raphael Loll-Krippleber, Grant W Brown, Jennifer A Surtees.

  Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate (dNTP) pools by ribonucleotide reductase (RNR) and repair of replication errors by the mismatch repair (MMR) system. We identified genetic interactions between rnr1 alleles that skew and/or elevate dNTP levels and MMR gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by MMR. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with MMR pathway defects. By combining rnr1 and msh mutations to alter and/or increase dNTP levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.

Keywords:  dNTP pools; deep sequencing; mismatch repair; mutation profiles; replication fidelity; ribonucleotide reductase

DOI:  https://doi.org/10.1093/genetics/iyac092
Front Oncol. 2022 ;12 904887

The Metabolic and Non-Metabolic Roles of UCK2 in Tumor Progression.

Yi Fu, Xin-Dong Wei, Luoting Guo, Kai Wu, Jiamei Le, Yujie Ma, Xiaoni Kong, Ying Tong, Hailong Wu.

  Enhanced nucleoside metabolism is one of the hallmarks of cancer. Uridine-cytidine kinase 2 (UCK2) is a rate-limiting enzyme of the pyrimidine salvage synthesis pathway to phosphorylate uridine and cytidine to uridine monophosphate (UMP) and cytidine monophosphate (CMP), respectively. Recent studies have shown that UCK2 is overexpressed in many types of solid and hematopoietic cancers, closely associates with poor prognosis, and promotes cell proliferation and migration in lung cancer and HCCs. Although UCK2 is thought to catalyze sufficient nucleotide building blocks to support the rapid proliferation of tumor cells, we and other groups have recently demonstrated that UCK2 may play a tumor-promoting role in a catalytic independent manner by activating oncogenic signaling pathways, such as STAT3 and EGFR-AKT. By harnessing the catalytic activity of UCK2, several cytotoxic ribonucleoside analogs, such as TAS-106 and RX-3117, have been developed for UCK2-mediated cancer chemotherapy. Moreover, we have demonstrated that the concurrent targeting of the catalytic dependent and independent features of UCK2 could synergistically inhibit tumor growth. These findings suggest that UCK2 may serve as a potential therapeutic target for cancer treatment. In this mini-review, we introduced the genomic localization and protein structure of UCK2, described the role of UCK2 in tumor development, discussed the application of UCK2 in anti-tumor treatment, and proposed concurrent targeting of the catalytic and non-catalytic roles of UCK2 as a potential therapeutic strategy for cancer treatment.

Keywords:  UCK2; chemotherapy; cytotoxic ribonucleoside analogs; non-metabolic role of UCK2; oncogene; pyrimidine salvage synthesis; tumor development and progression

DOI:  https://doi.org/10.3389/fonc.2022.904887
Mol Oncol. 2022 Jun 08.

The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.

Michael Lohmüller, Bernhard F Roeck, Tamas G Szabo, Marina Schapfl, Fragka Pegka, Sebastian Herzog, Andreas Villunger, Fabian Schuler.

  Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under-replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B-Cell CLL/Lymphoma 2 (BCL2)-regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53-deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome-wide CRISPR/Cas9-based loss-of-function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F-box protein S-phase Kinase-Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin-dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1-S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1-inhibitor treatment and reduced cell death in S-phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1-inhibitor treatment.

Keywords:  CHK1 inhibition; DNA-damage; SKP2; apoptosis; cell cycle; p27

DOI:  https://doi.org/10.1002/1878-0261.13264
Front Cell Dev Biol. 2022 ;10 884873

DNA Damage Response and Repair in Adaptive Immunity.

Sha Luo, Ruolin Qiao, Xuefei Zhang.

  The diversification of B-cell receptor (BCR), as well as its secreted product, antibody, is a hallmark of adaptive immunity, which has more specific roles in fighting against pathogens. The antibody diversification is from recombination-activating gene (RAG)-initiated V(D)J recombination, activation-induced cytidine deaminase (AID)-initiated class switch recombination (CSR), and V(D)J exon somatic hypermutation (SHM). The proper repair of RAG- and AID-initiated DNA lesions and double-strand breaks (DSBs) is required for promoting antibody diversification, suppressing genomic instability, and oncogenic translocations. DNA damage response (DDR) factors and DSB end-joining factors are recruited to the RAG- and AID-initiated DNA lesions and DSBs to coordinately resolve them for generating productive recombination products during antibody diversification. Recently, cohesin-mediated loop extrusion is proposed to be the underlying mechanism of V(D)J recombination and CSR, which plays essential roles in promoting the orientation-biased deletional end-joining . Here, we will discuss the mechanism of DNA damage repair in antibody diversification.

Keywords:  AID-initiated CSR and SHM; DNA damage repair; RAG-initiated V(D)J recombination; antibody diversification; cohesin-mediated loop extrusion

DOI:  https://doi.org/10.3389/fcell.2022.884873
Proc Natl Acad Sci U S A. 2022 Jun 07. 119(23): e2116462119

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

Lulu Bi, Zhenheng Qin, Teng Wang, Yanan Li, Xinshuo Jia, Xia Zhang, Xi-Miao Hou, Mauro Modesti, Xu-Guang Xi, Bo Sun.

  SignificanceBloom syndrome helicase (BLM) is a multifunctional helicase that primarily catalyzes the separation of two single strands of DNA. Here, using a single-molecule optical tweezers approach combined with confocal microscopy, we monitored both the enzymatic activity and oligomeric status of BLM at the same time. Strikingly, a head-on collision of BLM-medicated DNA unwinding forks was found to effectively switch their oligomeric state and activity. Specifically, BLMs, upon collision, immediately fuse across the fork junctions and covert their activities from dsDNA unwinding to ssDNA translocation and protein displacement. These findings explain how BLM plays multiple functional roles in homologous recombination (HR). The single-molecule approach used here provides a reference model for investigating the relationship between protein oligomeric state and function.

Keywords:  BLM; SSB; helicase; oligomerization; single molecule

DOI:  https://doi.org/10.1073/pnas.2116462119
J Biochem. 2022 Jun 03. pii: mvac042. [Epub ahead of print]

TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.

Hisao Masai.

  Stalled replication forks need to be swiftly detected, protected from collapse, and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3'-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA, and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Keywords:  3'-terminus binding/fork reversal; HLTF; PriA; stalled replication fork

DOI:  https://doi.org/10.1093/jb/mvac042
Nat Commun. 2022 Jun 08. 13(1): 3295

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Bertrand Theulot, Laurent Lacroix, Jean-Michel Arbona, Gael A Millot, Etienne Jean, Corinne Cruaud, Jade Pellet, Florence Proux, Magali Hennion, Stefan Engelen, Arnaud Lemainque, Benjamin Audit, Olivier Hyrien, Benoît Le Tallec.

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

DOI: https://doi.org/10.1038/s41467-022-31012-0
Cells. 2022 Jun 03. pii: 1832. [Epub ahead of print]11(11):

Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process.

Eunjin Ryu, Na Young Ha, Woojae Jung, Juyeong Yoo, Kyungjae Myung, Sukhyun Kang.

  Proliferating cell nuclear antigen (PCNA) is a DNA clamp that functions in key roles for DNA replication and repair. After the completion of DNA synthesis, PCNA should be unloaded from DNA in a timely way. The ATAD5-RFC-Like Complex (ATAD5-RLC) unloads PCNA from DNA. However, the mechanism of the PCNA-unloading process remains unclear. In this study, we determined the minimal PCNA-unloading domain (ULD) of ATAD5. We identified several motifs in the ATAD5 ULD that are essential in the PCNA-unloading process. The C-terminus of ULD is required for the stable association of RFC2-5 for active RLC formation. The N-terminus of ULD participates in the opening of the PCNA ring. ATAD5-RLC was more robustly bound to open-liable PCNA compared to the wild type. These results suggest that distinct motifs of the ATAD5 ULD participate in each step of the PCNA-unloading process.

Keywords:  ATAD5; PCNA; PCNA unloading; RFC; RFC-like complex; RLC; SUMOylation

DOI:  https://doi.org/10.3390/cells11111832
Biochem Biophys Res Commun. 2022 May 16. pii: S0006-291X(22)00745-8. [Epub ahead of print]615 9-16

Glycolysis addiction compensating for a defective pentose phosphate pathway confers gemcitabine sensitivity in SETD2-deficient pancreatic cancer.

Xuqing Shen, Yueyue Chen, Mingzhu Liu, Juanjuan Shi, Yingying Tang, Xiaotong Yang, Dapeng Xu, Hongfei Yao, Ping Lu, Yongwei Sun, Jing Xue, Ningning Niu.

  Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy driven by genetic mutations and/or epigenetic dysregulation. Gemcitabine chemotherapy is the first-line regimen for pancreatic cancer but has limited efficacy. Our previous study revealed the role of SETD2-H3K36me3 loss in the initiation and metastasis of PDAC, but little is known about its role in tumor metabolism. Here, we found that SETD2-deficient PDAC enhanced glycolysis addiction via upregulation of glucose transporter 1 (GLUT1) to meet its large demand for glucose in progression. Moreover, SETD2 deficiency impaired nucleoside synthesis by directly downregulating the transcriptional level of transketolase (TKT) in the pentose phosphate pathway. The metabolic changes confer SETD2-deficient PDAC cells with increased sensitivity to gemcitabine under glycolysis restriction conditions. Collectively, our study provides mechanistic insights into how SETD2 deficiency reprograms glycolytic metabolism to compensate for insufficient nucleoside synthesis, suggesting that glycolysis restriction combined with gemcitabine might be a potential therapeutic strategy for PDAC patients with SETD2 deficiency.

Keywords:  GLUT1; Metabolic reprogramming; PDAC; SETD2; TKT

DOI:  https://doi.org/10.1016/j.bbrc.2022.05.047
Commun Biol. 2022 Jun 09. 5(1): 571

TAS1553, a small molecule subunit interaction inhibitor of ribonucleotide reductase, exhibits antitumor activity by causing DNA replication stress.

Hiroyuki Ueno, Takuya Hoshino, Wakako Yano, Sayaka Tsukioka, Takamasa Suzuki, Shoki Hara, Yoshio Ogino, Khoon Tee Chong, Tatsuya Suzuki, Shingo Tsuji, Hikaru Itadani, Ikuo Yamamiya, Yoshihiro Otsu, Satoshi Ito, Toshiya Yonekura, Miki Terasaka, Nozomu Tanaka, Seiji Miyahara.

Ribonucleotide reductase (RNR) is composed of two non-identical subunits, R1 and R2, and plays a crucial role in balancing the cellular dNTP pool, establishing it as an attractive cancer target. Herein, we report the discovery of a highly potent and selective small-molecule inhibitor, TAS1553, targeting protein-protein interaction between R1 and R2. TAS1553 is also expected to demonstrate superior selectivity because it does not directly target free radical or a substrate binding site. TAS1553 has shown antiproliferative activity in human cancer cell lines, dramatically reducing the intracellular dATP pool and causing DNA replication stress. Furthermore, we identified SLFN11 as a biomarker that predicts the cytotoxic effect of TAS1553. Oral administration of TAS1553 demonstrated robust antitumor efficacy against both hematological and solid cancer xenograft tumors and also provided a significant survival benefit in an acute myelogenous leukemia model. Our findings strongly support the evaluation of TAS1553 in clinical trials.

DOI: https://doi.org/10.1038/s42003-022-03516-4
Mol Cell. 2022 May 25. pii: S1097-2765(22)00444-0. [Epub ahead of print]

Condensates induced by transcription inhibition localize active chromatin to nucleoli.

Takaaki Yasuhara, Yu-Hang Xing, Nicholas C Bauer, Lukuo Lee, Rui Dong, Tribhuwan Yadav, Roy J Soberman, Miguel N Rivera, Lee Zou.

  The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

Keywords:  chromatin; compartment; gene fusion; nucleolus; phase separation; rRNA; transcription

DOI:  https://doi.org/10.1016/j.molcel.2022.05.010
Sci Rep. 2022 Jun 10. 12(1): 9560

Telomere-to-telomere human DNA replication timing profiles.

Dashiell J Massey, Amnon Koren.

The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.

DOI: https://doi.org/10.1038/s41598-022-13638-8
Cancers (Basel). 2022 Jun 05. pii: 2804. [Epub ahead of print]14(11):

Insights into the Possible Molecular Mechanisms of Resistance to PARP Inhibitors.

Claudia Piombino, Laura Cortesi.

  PARP1 enzyme plays an important role in DNA damage recognition and signalling. PARP inhibitors are approved in breast, ovarian, pancreatic, and prostate cancers harbouring a pathogenic variant in BRCA1 or BRCA2, where PARP1 inhibition results mainly in synthetic lethality in cells with impaired homologous recombination. However, the increasingly wide use of PARP inhibitors in clinical practice has highlighted the problem of resistance to therapy. Several different mechanisms of resistance have been proposed, although only the acquisition of secondary mutations in BRCA1/2 has been clinically proved. The aim of this review is to outline the key molecular findings that could explain the development of primary or secondary resistance to PARP inhibitors, analysing the complex interactions between PARP1, cell cycle regulation, PI3K/AKT signalling, response to stress replication, homologous recombination, and other DNA damage repair pathways in the setting of BRCA1/2 mutated cancers.

Keywords:  BRCA1; BRCA2; PARP inhibitors; fork stabilization; homologous recombination; non-homologous end joining

DOI:  https://doi.org/10.3390/cancers14112804
Oncogene. 2022 Jun 03.

Functional RECAP (REpair CAPacity) assay identifies homologous recombination deficiency undetected by DNA-based BRCAness tests.

Titia G Meijer, Luan Nguyen, Arne Van Hoeck, Anieta M Sieuwerts, Nicole S Verkaik, Marjolijn M Ladan, Kirsten Ruigrok-Ritstier, Carolien H M van Deurzen, Harmen J G van de Werken, Esther H Lips, Sabine C Linn, Yasin Memari, Helen Davies, Serena Nik-Zainal, Roland Kanaar, John W M Martens, Edwin Cuppen, Agnes Jager, Dik C van Gent.

Germline BRCA1/2 mutation status is predictive for response to Poly-[ADP-Ribose]-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated and homologous recombination repair deficient (HRD) tumors are likely also PARP-inhibitor sensitive. Clinical validity and utility of various HRD biomarkers are under investigation. The REpair CAPacity (RECAP) test is a functional method to select HRD tumors based on their inability to form RAD51 foci. We investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests. An HRD enriched cohort (n = 71; 52 primary and 19 metastatic BCs) selected based on the RECAP test (26 RECAP-HRD; 37%), was subjected to DNA-based HRD tests (i.e., Classifier of HOmologous Recombination Deficiency (CHORD) and BRCA1/2-like classifier). Whole genome sequencing (WGS) was carried out for 38 primary and 19 metastatic BCs. The RECAP test identified all bi-allelic BRCA deficient samples (n = 15) in this cohort. RECAP status partially correlated with DNA-based HRD test outcomes (70% concordance for both RECAP-CHORD and RECAP-BRCA1/2-like classifier). RECAP selected additional samples unable to form RAD51 foci, suggesting that this functional assay identified deficiencies in other DNA repair genes, which could also result in PARP-inhibitor sensitivity. Direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making.

DOI: https://doi.org/10.1038/s41388-022-02363-1
Sci Adv. 2022 Jun 03. 8(22): eabn3815

Concordance of hydrogen peroxide-induced 8-oxo-guanine patterns with two cancer mutation signatures of upper GI tract tumors.

Seung-Gi Jin, Yingying Meng, Jennifer Johnson, Piroska E Szabó, Gerd P Pfeifer.

Oxidative DNA damage has been linked to inflammation, cancer, and aging. Here, we have mapped two types of oxidative DNA damage, oxidized guanines produced by hydrogen peroxide and oxidized thymines created by potassium permanganate, at a single-base resolution. 8-Oxo-guanine occurs strictly dependent on the G/C sequence context and shows a pronounced peak at transcription start sites (TSSs). We determined the trinucleotide sequence pattern of guanine oxidation. This pattern shows high similarity to the cancer-associated single-base substitution signatures SBS18 and SBS36. SBS36 is found in colorectal cancers that carry mutations in MUTYH, encoding a repair enzyme that operates on 8-oxo-guanine mispairs. SBS18 is common in inflammation-associated upper gastrointestinal tract tumors including esophageal and gastric adenocarcinomas. Oxidized thymines induced by permanganate occur with a distinct dinucleotide specificity, 5'T-A/C, and are depleted at the TSS. Our data suggest that two cancer mutational signatures, SBS18 and SBS36, are caused by reactive oxygen species.

DOI: https://doi.org/10.1126/sciadv.abn3815
Sci Adv. 2022 Jun 10. 8(23): eabn7063

Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks.

Kristian Jeppsson, Toyonori Sakata, Ryuichiro Nakato, Stefina Milanova, Katsuhiko Shirahige, Camilla Björkegren.

Genome function depends on regulated chromosome folding, and loop extrusion by the protein complex cohesin is essential for this multilayered organization. The chromosomal positioning of cohesin is controlled by transcription, and the complex also localizes to stalled replication forks. However, the role of transcription and replication in chromosome looping remains unclear. Here, we show that reduction of chromosome-bound RNA polymerase weakens normal cohesin loop extrusion boundaries, allowing cohesin to form new long-range chromosome cis interactions. Stress response genes induced by transcription inhibition are also shown to act as new loop extrusion boundaries. Furthermore, cohesin loop extrusion during early S phase is jointly controlled by transcription and replication units. Together, the results reveal that replication and transcription machineries are chromosome-folding regulators that block the progression of loop-extruding cohesin, opening for new perspectives on cohesin's roles in genome function and stability.

DOI: https://doi.org/10.1126/sciadv.abn7063
Arch Microbiol. 2022 Jun 10. 204(7): 383

Control of a pyrimidine ribonucleotide salvage pathway in Pseudomonas oleovorans.

Ramneetpreet Gill, Thomas P West.

  The control of a pyrimidine ribonucleotide salvage pathway in the bacterium Pseudomonas oleovorans ATCC 8062 was studied. This bacterium is important for its ability to synthesize polyesters as well as for its increasing clinical significance in humans. The pyrimidine salvage pathway enzymes pyrimidine nucleotide N-ribosidase and cytosine deaminase were investigated in P. oleovorans ATCC 8062 under selected culture conditions. Initially, the effect of carbon source on the two pyrimidine salvage enzymes in ATCC 8062 cells was examined and it was observed that cell growth on the carbon source succinate generally produced higher enzyme activities than did glucose or glycerol as a carbon source when ammonium sulfate served as the nitrogen source. Using succinate as a carbon source, growth on dihydrouracil as nitrogen source caused a 1.9-fold increase in the pyrimidine nucleotide N-ribosidase activity and a 4.8-fold increase in cytosine deaminase activity compared to the ammonium sulfate-grown cells. Growth of ATCC 8062 cells on cytosine or dihydrothymine as a nitrogen source elevated deaminase activity by more than double that observed for ammonium sulfate-grown cells. The findings indicated a relationship between this pyrimidine salvage pathway and the pyrimidine reductive catabolic pathway since growth on dihydrouracil appeared to increase the degradation of the pyrimidine ribonucleotide monophosphates to uracil. The uracil produced could be degraded by the pyrimidine base reductive catabolic pathway to β-alanine as a source of nitrogen. This investigation could prove helpful to future work examining the metabolic relationship between pyrimidine salvage pathways and pyrimidine reductive catabolism in pseudomonads.

Keywords:  Cytosine deaminase; Pseudomonas oleovorans; Pyrimidine ribonucleotide N-ribosidase; Pyrimidine salvage; Regulation

DOI:  https://doi.org/10.1007/s00203-022-03016-3
Nature. 2022 Jun 08.

Cohesin-mediated loop anchors confine the locations of human replication origins.

Daniel J Emerson, Peiyao A Zhao, Ashley L Cook, R Jordan Barnett, Kyle N Klein, Dalila Saulebekova, Chunmin Ge, Linda Zhou, Zoltan Simandi, Miriam K Minsk, Katelyn R Titus, Weitao Wang, Wanfeng Gong, Di Zhang, Liyan Yang, Sergey V Venev, Johan H Gibcus, Hongbo Yang, Takayo Sasaki, Masato T Kanemaki, Feng Yue, Job Dekker, Chun-Long Chen, David M Gilbert, Jennifer E Phillips-Cremins.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

DOI: https://doi.org/10.1038/s41586-022-04803-0
Nature. 2022 Jun 08.

Core control principles of the eukaryotic cell cycle.

Souradeep Basu, Jessica Greenwood, Andrew W Jones, Paul Nurse.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

DOI: https://doi.org/10.1038/s41586-022-04798-8
Cancers (Basel). 2022 May 24. pii: 2580. [Epub ahead of print]14(11):

Targeting MUC1-C Suppresses Chronic Activation of Cytosolic Nucleotide Receptors and STING in Triple-Negative Breast Cancer.

Nami Yamashita, Atsushi Fushimi, Yoshihiro Morimoto, Atrayee Bhattacharya, Masayuki Hagiwara, Masaaki Yamamoto, Tsuyoshi Hata, Geoffrey I Shapiro, Mark D Long, Song Liu, Donald Kufe.

  The MUC1-C apical transmembrane protein is activated in the acute response of epithelial cells to inflammation. However, chronic MUC1-C activation promotes cancer progression, emphasizing the importance of MUC1-C as a target for treatment. We report here that MUC1-C is necessary for intrinsic expression of the RIG-I, MDA5 and cGAS cytosolic nucleotide pattern recognition receptors (PRRs) and the cGAS-stimulator of IFN genes (STING) in triple-negative breast cancer (TNBC) cells. Consistent with inducing the PRR/STING axis, MUC1-C drives chronic IFN-β production and activation of the type I interferon (IFN) pathway. MUC1-C thereby induces the IFN-related DNA damage resistance gene signature (IRDS), which includes ISG15, in linking chronic inflammation with DNA damage resistance. Targeting MUC1-C in TNBC cells treated with carboplatin or the PARP inhibitor olaparib further demonstrated that MUC1-C is necessary for expression of PRRs, STING and ISG15 and for intrinsic DNA damage resistance. Of translational relevance, MUC1 significantly associates with upregulation of STING and ISG15 in TNBC tumors and is a target for treatment with CAR T cells, antibody-drug conjugates (ADCs) and direct inhibitors that are under preclinical and clinical development.

Keywords:  ISG15; MUC1-C; STING; TNBC; cGAS

DOI:  https://doi.org/10.3390/cancers14112580
Curr Res Struct Biol. 2022 ;4 192-205

Insight into the nucleoside transport and inhibition of human ENT1.

Zhixiang Wu, Zhongjie Han, Wenxue Zhou, Xiaohan Sun, Lei Chen, Shuang Yang, Jianping Hu, Chunhua Li.

  The human equilibrative nucleoside transporter 1 (hENT1) is an effective controller of adenosine signaling by regulating its extracellular and intracellular concentration, and has become a solid drug target of clinical used adenosine reuptake inhibitors (AdoRIs). Currently, the mechanisms of adenosine transport and inhibition for hENT1 remain unclear, which greatly limits the in-depth understanding of its inner workings as well as the development of novel inhibitors. In this work, the dynamic details of hENT1 underlie adenosine transport and the inhibition mechanism of the non-nucleoside AdoRIs dilazep both were investigated by comparative long-time unbiased molecular dynamics simulations. The calculation results show that the conformational transitions of hENT1 from the outward open to metastable occluded state are mainly driven by TM1, TM2, TM7 and TM9. One of the trimethoxyphenyl rings in dilazep serves as the adenosyl moiety of the endogenous adenosine substrate to competitively occupy the orthosteric site of hENT1. Due to extensive and various VDW interactions with N30, M33, M84, P308 and F334, the other trimethoxyphenyl ring is stuck in the opportunistic site near the extracellular side preventing the complete occlusion of thin gate simultaneously. Obviously, dilazep shows significant inhibitory activity by disrupting the local induce-fit action in substrate binding cavity and blocking the transport cycle of whole protein. This study not only reveals the nucleoside transport mechanism by hENT1 at atomic level, but also provides structural guidance for the subsequent design of novel non-nucleoside AdoRIs with enhanced pharmacologic properties.

Keywords:  Adenosine; Dilazep; Inhibition mechanism; Nucleoside transport; hENT1

DOI:  https://doi.org/10.1016/j.crstbi.2022.05.005
Cell Cycle. 2022 Jun 07. 1-14

SAMHD1, positively regulated by KLF4, suppresses the proliferation of gastric cancer cells through MAPK p38 signaling pathway.

Zhangming Chen, Zhe Jiang, Lei Meng, Ye Wang, Minggui Lin, Zhijian Wei, Wenxiu Han, Songcheng Ying, Aman Xu.

  SAMHD1 was reported to be related with the development of tumors, while its function in gastric cancer (GC) has not been elucidated yet. Here, we investigated the role and mechanism of SAMHD1 in regulating the proliferation of GC, as well as the mechanism of its expression regulation. Our results revealed that SAMHD1 was downregulated in GC tissues and cell lines, which was correlated with tumor size, depth of invasion and TNM stage. Overexpression of SAMHD1 inhibited the proliferation, clone formation, DNA synthesis and cell cycle progression, while knockdown of SAMHD1 promoted the proliferation of GC cells in vitro and vivo. Meanwhile, SAMHD1 inhibited the activation of MAPK p38 signaling pathway. Moreover, SB203580, as a MAPK p38 inhibitor, could reverse the proliferation and activation of MAPK p38 signaling pathway caused by knockdown of SAMHD1 in GC cells. Additionally, transcription factor Krüppel-like factor 4 (KLF4) bound to the core promoter of SAMHD1, increasing its transcriptional expression in GC cells. In conclusion, SAMHD1 suppressed the proliferation of GC through negatively regulating the activation of MAPK p38 signaling pathway and was upregulated by KLF4 in GC cells.

Keywords:  Gastric cancer; KLF4; MAPK p38; SAMHD1; proliferation

DOI:  https://doi.org/10.1080/15384101.2022.2085356
ACS Chem Biol. 2022 Jun 09.

Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Šimon Pospíšil, Alessandro Panattoni, Filip Gracias, Veronika Sýkorová, Viola Vaňková Hausnerová, Dragana Vítovská, Hana Šanderová, Libor Krásný, Michal Hocek.

Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

DOI: https://doi.org/10.1021/acschembio.2c00342