bims-numges 2021-08-15 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–08–15
34 papers selected by
Sean Rudd, Karolinska Institutet

The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.
The PRIMPOL Protein Shields Replication Forks in BRCA-Deficient Cells.
A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes.
MTAP deficiency-induced metabolic reprogramming creates a vulnerability to co-targeting de novo purine synthesis and glycolysis in pancreatic cancer.
RECQ1 Promotes Stress Resistance and DNA Replication Progression Through PARP1 Signaling Pathway in Glioblastoma.
hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors.
Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.
How DNA damage and non-canonical nucleotides alter the telomerase catalytic cycle.
Implications of inhibition of Rev1 interaction with Y family DNA polymerases for cisplatin chemotherapy.
CometChip enables parallel analysis of multiple DNA repair activities.
A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells.
Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini.
RepairSig: Deconvolution of DNA damage and repair contributions to the mutational landscape of cancer.
Fused in sarcoma (FUS) regulates DNA replication timing and kinetics.
Genotoxic stress and viral infection induce transient expression of APOBEC3A and pro-inflammatory genes through two distinct pathways.
Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex.
The Fate of Two Unstoppable Trains After Arriving Destination: Replisome Disassembly During DNA Replication Termination.
Targeting the DNA damage response in immuno-oncology: developments and opportunities.
CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.
The alarmone (p)ppGpp regulates primer extension by bacterial primase.
Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition.
Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.
Inhibition of the DSB repair protein RAD51 potentiates the cytotoxic efficacy of doxorubicin via promoting apoptosis-related death pathways.
Cancer cells are highly susceptible to accumulation of templated insertions linked to MMBIR.
Relapse-Driving AML Cells Exhibit Pyrimidine-Synthesis Dependency.
DNA replication: the recombination connection.
Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.
A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family.
Mechanism of chromosome rearrangement arising from single-strand breaks.
Histone Methylation Connects DNA Repair to Metabolism in Cancer Cells.
Different DNA repair pathways are involved in single-strand break-induced genomic changes in plants.
Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage.
Recombination-dependent replication: new perspectives from site-specific fork barriers.
A feedback loop between the androgen receptor and 6-phosphogluoconate dehydrogenase (6PGD) drives prostate cancer growth.

Genes Dev. 2021 Aug 12.

The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.

Antoine Simoneau, Rosalinda Xiong, Lee Zou.

  PARP inhibitor (PARPi) is widely used to treat BRCA1/2-deficient tumors, but why PARPi is more effective than other DNA-damaging drugs is unclear. Here, we show that PARPi generates DNA double-strand breaks (DSBs) predominantly in a trans cell cycle manner. During the first S phase after PARPi exposure, PARPi induces single-stranded DNA (ssDNA) gaps behind DNA replication forks. By trapping PARP on DNA, PARPi prevents the completion of gap repair until the next S phase, leading to collisions of replication forks with ssDNA gaps and a surge of DSBs. In the second S phase, BRCA1/2-deficient cells are unable to suppress origin firing through ATR, resulting in continuous DNA synthesis and more DSBs. Furthermore, BRCA1/2-deficient cells cannot recruit RAD51 to repair collapsed forks. Thus, PARPi induces DSBs progressively through trans cell cycle ssDNA gaps, and BRCA1/2-deficient cells fail to slow down and repair DSBs over multiple cell cycles, explaining the unique efficacy of PARPi in BRCA1/2-deficient cells.

Keywords:  BRCA; DNA damage; PARP inhibitor; cell cycle; replication

DOI:  https://doi.org/10.1101/gad.348479.121
Cancer Discov. 2020 Jan;10(1): 14

The PRIMPOL Protein Shields Replication Forks in BRCA-Deficient Cells.

PRIMPOL shields replication forks after multiple rounds of cisplatin in BRCA-deficient cancer cells.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2019-168
Nucleic Acids Res. 2021 Aug 09. pii: gkab663. [Epub ahead of print]

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes.

Vivek M Shastri, Veena Subramanian, Kristina H Schmidt.

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.

DOI: https://doi.org/10.1093/nar/gkab663
Cancer Res. 2021 Aug 12. pii: canres.0414.2020. [Epub ahead of print]

MTAP deficiency-induced metabolic reprogramming creates a vulnerability to co-targeting de novo purine synthesis and glycolysis in pancreatic cancer.

Qiangsheng Hu, Yi Qin, Shunrong Ji, Xiuhui Shi, Weixing Dai, Guixiong Fan, Shuo Li, Wenyan Xu, Wensheng Liu, Mengqi Liu, Zheng Zhang, Zeng Ye, Zhijun Zhou, Jingxuan Yang, Qifeng Zhuo, Xianjun Yu, Min Li, Xiaowu Xu.

Methylthioadenosine phosphorylase (MTAP) is a key enzyme associated with the salvage of methionine and adenine that is deficient in 20%-30% of pancreatic cancer. Our previous study revealed that MTAP-deficiency indicates a poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients. In this study, bioinformatics analysis of The Cancer Genome Atlas (TCGA) data indicated that PDACs with MTAP deficiency display a signature of elevated glycolysis. Metabolomics studies showed that that MTAP deletion-mediated metabolic reprogramming enhanced glycolysis and de novo purine synthesis in pancreatic cancer cells. Western blot analysis revealed that MTAP knockout stabilized hypoxia-inducible factor 1α (HIF-1α) protein via posttranslational phosphorylation. RIO kinase 1 (RIOK1), a downstream kinase upregulated in MTAP-deficient cells, interacted with and phosphorylated HIF-1α to regulate its stability. In vitro experiments demonstrated that the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) and the de novo purine synthesis inhibitor L-alanosine synergized to kill MTAP-deficient pancreatic cancer cells. Collectively, these results reveal that MTAP deficiency drives pancreatic cancer progression by inducing metabolic reprogramming, providing a novel target and therapeutic strategy for treating MTAP-deficient disease.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-0414
Front Cell Dev Biol. 2021 ;9 714868

RECQ1 Promotes Stress Resistance and DNA Replication Progression Through PARP1 Signaling Pathway in Glioblastoma.

Jing Zhang, Hao Lian, Kui Chen, Ying Pang, Mu Chen, Bingsong Huang, Lei Zhu, Siyi Xu, Min Liu, Chunlong Zhong.

  Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor, and patients with GBM have a median survival of 20 months. Clinical therapy resistance is a challenging barrier to overcome. Tumor genome stability maintenance during DNA replication, especially the ability to respond to replication stress, is highly correlated with drug resistance. Recently, we identified a protective role for RECQ1 under replication stress conditions. RECQ1 acts at replication forks, binds PCNA, inhibits single-strand DNA formation and nascent strand degradation in GBM cells. It is associated with the function of the PARP1 protein, promoting PARP1 recruitment to replication sites. RECQ1 is essential for DNA replication fork protection and tumor cell proliferation under replication stress conditions, and as a target of RECQ1, PARP1 effectively protects and restarts stalled replication forks, providing new insights into genomic stability maintenance and replication stress resistance. These findings indicate that tumor genome stability targeting RECQ1-PARP1 signaling may be a promising therapeutic intervention to overcome therapy resistance in GBM.

Keywords:  DNA replication stress; PARP1; RECQ1; drug resistance; fork reversal; genomic stability

DOI:  https://doi.org/10.3389/fcell.2021.714868
Cell Signal. 2021 Aug 04. pii: S0898-6568(21)00195-9. [Epub ahead of print] 110106

hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors.

Ramazan Gundogdu, M Kadir Erdogan, Angeliki Ditsiou, Victoria Spanswick, Juan Jose Garcia Gomez, John A Hartley, Fumiko Esashi, Alexander Hergovich, Valenti Gomez.

  Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.

Keywords:  DNA damage response signalling; Homologous recombination DNA repair; Mps one binder 2 (MOB2); Personalised PARP inhibitor treatments

DOI:  https://doi.org/10.1016/j.cellsig.2021.110106
Elife. 2021 Aug 13. pii: e69726. [Epub ahead of print]10

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

Allison McClure, John Diffley.

  The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Keywords:  S. cerevisiae; chromosomes; gene expression

DOI:  https://doi.org/10.7554/eLife.69726
DNA Repair (Amst). 2021 Jul 31. pii: S1568-7864(21)00154-3. [Epub ahead of print]107 103198

How DNA damage and non-canonical nucleotides alter the telomerase catalytic cycle.

Samantha L Sanford, Griffin A Welfer, Bret D Freudenthal, Patricia L Opresko.

  Telomeres at the ends of linear chromosomes are essential for genome maintenance and sustained cellular proliferation, but shorten with each cell division. Telomerase, a specialized reverse transcriptase with its own integral RNA template, compensates for this by lengthening the telomeric 3' single strand overhang. Mammalian telomerase has the unique ability to processively synthesize multiple GGTTAG repeats, by translocating along its product and reiteratively copying the RNA template, termed repeat addition processivity (RAP). This unusual form of processivity is distinct from the nucleotide addition processivity (NAP) shared by all other DNA polymerases. In this review, we focus on the minimally active human telomerase catalytic core consisting of the telomerase reverse transcriptase (TERT) and the integral RNA (TR), which catalyzes DNA synthesis. We review the mechanisms by which oxidatively damaged nucleotides, and anti-viral and anti-cancer nucleotide drugs affect the telomerase catalytic cycle. Finally, we offer perspective on how we can leverage telomerase's unique properties, and advancements in understanding of telomerase catalytic mechanism, to selectively manipulate telomerase activity with therapeutics, particularly in cancer treatment.

Keywords:  Aging; Cancer; DNA damage; DNA repair; Nucleoside analogs; Ribonucleotides; Telomerase; Telomerase therapeutics; Telomeres

DOI:  https://doi.org/10.1016/j.dnarep.2021.103198
Genes Dev. 2021 Aug 12.

Implications of inhibition of Rev1 interaction with Y family DNA polymerases for cisplatin chemotherapy.

Jung-Hoon Yoon, Robert E Johnson, Louise Prakash, Satya Prakash.

  Chemotherapy with cisplatin becomes limiting due to toxicity and secondary malignancies. In principle, therapeutics could be improved by targeting translesion synthesis (TLS) polymerases (Pols) that promote replication through intrastrand cross-links, the major cisplatin-induced DNA adduct. However, to specifically target malignancies with minimal adverse effects on normal cells, a good understanding of TLS mechanisms in normal versus cancer cells is paramount. We show that in normal cells, TLS through cisplatin intrastrand cross-links is promoted by Polη- or Polι-dependent pathways, both of which require Rev1 as a scaffolding component. In contrast, cancer cells require Rev1-Polζ. Our findings that a recently identified Rev1 inhibitor, JH-RE-06, purported to specifically disrupt Rev1 interaction with Polζ to block TLS through cisplatin adducts in cancer cells, abrogates Rev1's ability to function with Y family Pols as well, implying that by inactivating Rev1-dependent TLS in normal cells, this inhibitor will exacerbate the toxicity and tumorigenicity of chemotherapeutics with cisplatin.

Keywords:  DNA repair; Rev1; Y family DNA polymerases; cisplatin intrastrand cross-links; nucleotide excision repair; translesion synthesis

DOI:  https://doi.org/10.1101/gad.348662.121
DNA Repair (Amst). 2021 Jul 10. pii: S1568-7864(21)00132-4. [Epub ahead of print]106 103176

CometChip enables parallel analysis of multiple DNA repair activities.

Jing Ge, Le P Ngo, Simran Kaushal, Ian J Tay, Elina Thadhani, Jennifer E Kay, Patrizia Mazzucato, Danielle N Chow, Jessica L Fessler, David M Weingeist, Robert W Sobol, Leona D Samson, Scott R Floyd, Bevin P Engelward.

  DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

Keywords:  Comet assay; CometChip; DNA damage; DNA repair

DOI:  https://doi.org/10.1016/j.dnarep.2021.103176
Nat Commun. 2021 Aug 13. 12(1): 4919

A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells.

Rebecca A Dagg, Gijs Zonderland, Emilia Puig Lombardi, Giacomo G Rossetti, Florian J Groelly, Sonia Barroso, Eliana M C Tacconi, Benjamin Wright, Helen Lockstone, Andrés Aguilera, Thanos D Halazonetis, Madalena Tarsounas.

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

DOI: https://doi.org/10.1038/s41467-021-25215-0
Nat Commun. 2021 08 10. 12(1): 4843

Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini.

Joost Schimmel, Núria Muñoz-Subirana, Hanneke Kool, Robin van Schendel, Marcel Tijsterman.

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

DOI: https://doi.org/10.1038/s41467-021-25154-w
Cell Syst. 2021 Aug 05. pii: S2405-4712(21)00282-9. [Epub ahead of print]

RepairSig: Deconvolution of DNA damage and repair contributions to the mutational landscape of cancer.

Damian Wojtowicz, Jan Hoinka, Bayarbaatar Amgalan, Yoo-Ah Kim, Teresa M Przytycka.

  Cancer genomes accumulate a large number of somatic mutations resulting from a combination of stochastic errors in DNA processing, cancer-related aberrations of the DNA repair machinery, or carcinogenic exposures; each mutagenic process leaves a characteristic mutational signature. A key challenge is understanding the interactions between signatures, particularly as DNA repair deficiencies often modify the effects of other mutagens. Here, we introduce RepairSig, a computational method that explicitly models additive primary mutagenic processes; non-additive secondary processes, which interact with the primary processes; and a mutation opportunity, that is, the distribution of sites across the genome that are vulnerable to damage or preferentially repaired. We demonstrate that RepairSig accurately recapitulates experimentally identified signatures, identifies autonomous signatures of deficient DNA repair processes, and explains mismatch repair deficiency in breast cancer by de novo inference of both primary and secondary signatures from patient data. RepairSig is freely available for download at https://github.com/ncbi/RepairSig.

Keywords:  DNA damage; DNA mismatch repair; DNA repair; cancer; mutational signatures; tensorflow

DOI:  https://doi.org/10.1016/j.cels.2021.07.004
J Biol Chem. 2021 Aug 07. pii: S0021-9258(21)00851-6. [Epub ahead of print] 101049

Fused in sarcoma (FUS) regulates DNA replication timing and kinetics.

Weiyan Jia, Sang Hwa Kim, Mark A Scalf, Peter Tonzi, Robert J Millikin, William M Guns, Lu Liu, Adam S Mastrocola, Lloyd M Smith, Tony T Huang, Randal S Tibbetts.

Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis (ALS). FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break (DSB) repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of DSB-associated factors, including 53BP1 and BRCA1. FUS-/- cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of pre-replication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase-associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS cDNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.

DOI: https://doi.org/10.1016/j.jbc.2021.101049
Nat Commun. 2021 Aug 13. 12(1): 4917

Genotoxic stress and viral infection induce transient expression of APOBEC3A and pro-inflammatory genes through two distinct pathways.

Sunwoo Oh, Elodie Bournique, Danae Bowen, Pégah Jalili, Ambrocio Sanchez, Ian Ward, Alexandra Dananberg, Lavanya Manjunath, Genevieve P Tran, Bert L Semler, John Maciejowski, Marcus Seldin, Rémi Buisson.

APOBEC3A is a cytidine deaminase driving mutagenesis in tumors. While APOBEC3A-induced mutations are common, APOBEC3A expression is rarely detected in cancer cells. This discrepancy suggests a tightly controlled process to regulate episodic APOBEC3A expression in tumors. In this study, we find that both viral infection and genotoxic stress transiently up-regulate APOBEC3A and pro-inflammatory genes using two distinct mechanisms. First, we demonstrate that STAT2 promotes APOBEC3A expression in response to foreign nucleic acid via a RIG-I, MAVS, IRF3, and IFN-mediated signaling pathway. Second, we show that DNA damage and DNA replication stress trigger a NF-κB (p65/IkBα)-dependent response to induce expression of APOBEC3A and other innate immune genes, independently of DNA or RNA sensing pattern recognition receptors and the IFN-signaling response. These results not only reveal the mechanisms by which tumors could episodically up-regulate APOBEC3A but also highlight an alternative route to stimulate the immune response after DNA damage independently of cGAS/STING or RIG-I/MAVS.

DOI: https://doi.org/10.1038/s41467-021-25203-4
Nucleic Acids Res. 2021 Aug 14. pii: gkab646. [Epub ahead of print]

Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex.

Siqi Shen, Gregory A Davidson, Kun Yang, Zhihao Zhuang.

The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.

DOI: https://doi.org/10.1093/nar/gkab646
Front Cell Dev Biol. 2021 ;9 658003

The Fate of Two Unstoppable Trains After Arriving Destination: Replisome Disassembly During DNA Replication Termination.

Yisui Xia.

  In eukaryotes, the perfect duplication of the chromosomes is executed by a dynamic molecular machine called the replisome. As a key step to finishing DNA replication, replisome disassembly is triggered by ubiquitylation of the MCM7 subunit of the helicase complex CMG. Afterwards, the CDC48/p97 "unfoldase" is recruited to the ubiquitylated helicase to unfold MCM7 and disassemble the replisome. Here we summarise recently discovered mechanisms of replisome disassembly that are likely to be broadly conserved in eukaryotes. We also discuss two crucial questions that remain to be explored further in the future. Firstly, how is CMG ubiquitylation repressed by the replication fork throughout elongation? Secondly, what is the biological significance of replisome disassembly and what are the consequences of failing to ubiquitylate and disassemble the CMG helicase?

Keywords:  CMG; CUL2cpsdummyLRR1; SCFcpsdummyDia2; p97/CDC48/VCP; replisome disassembly; ubiquitylation

DOI:  https://doi.org/10.3389/fcell.2021.658003
Nat Rev Cancer. 2021 Aug 10.

Targeting the DNA damage response in immuno-oncology: developments and opportunities.

Roman M Chabanon, Mathieu Rouanne, Christopher J Lord, Jean-Charles Soria, Philippe Pasero, Sophie Postel-Vinay.

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcome with regard to multiple tumour types. However, most patients still do not benefit from such therapies, notably because of the absence of pre-existing T cell infiltration. DNA damage response (DDR) deficiency has recently emerged as an important determinant of tumour immunogenicity. A growing body of evidence now supports the concept that DDR-targeted therapies can increase the antitumour immune response by (1) promoting antigenicity through increased mutability and genomic instability, (2) enhancing adjuvanticity through the activation of cytosolic immunity and immunogenic cell death and (3) favouring reactogenicity through the modulation of factors that control the tumour-immune cell synapse. In this Review, we discuss the interplay between the DDR and anticancer immunity and highlight how this dynamic interaction contributes to shaping tumour immunogenicity. We also review the most innovative preclinical approaches that could be used to investigate such effects, including recently developed ex vivo systems. Finally, we highlight the therapeutic opportunities presented by the exploitation of the DDR-anticancer immunity interplay, with a focus on those in early-phase clinical development.

DOI: https://doi.org/10.1038/s41568-021-00386-6
J Cell Sci. 2021 Aug 01. pii: jcs258601. [Epub ahead of print]134(15):

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Swati Priya, Ekjot Kaur, Swati Kulshrestha, Awadhesh Pandit, Isabelle Gross, Nitin Kumar, Himanshi Agarwal, Aamir Khan, Radhey Shyam, Prakash Bhagat, Jyothi S Prabhu, Perumal Nagarajan, S V S Deo, Avinash Bajaj, Jean-Noël Freund, Arnab Mukhopadhyay, Sagar Sengupta.

  Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

Keywords:  ATM; BLM; BRCA1; Chk1; Colon cancer; DNA damage response; DNA repair; RNF8; microRNA

DOI:  https://doi.org/10.1242/jcs.258601
J Mol Biol. 2021 Aug 10. pii: S0022-2836(21)00422-8. [Epub ahead of print] 167189

The alarmone (p)ppGpp regulates primer extension by bacterial primase.

Christina N Giramma, McKenna B DeFoer, Jue D Wang.

  Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.

Keywords:  (p)ppGpp; bacterial primase; genome stability; regulation of DNA replication; the stringent response

DOI:  https://doi.org/10.1016/j.jmb.2021.167189
Neoplasia. 2021 Aug 08. pii: S1476-5586(21)00051-8. [Epub ahead of print]23(9): 1002-1015

Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition.

Chinnadurai Mani, Kaushlendra Tripathi, Sandeep Chaudhary, Ranganatha R Somasagara, Rodney P Rocconi, Chiquito Crasto, Mark Reedy, Mohammad Athar, Komaraiah Palle.

  Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses.

Keywords:  FANCD2; GANT61; GLI1; Olaparib; Ovarian Cancer; and Homologous recombination deficiency

DOI:  https://doi.org/10.1016/j.neo.2021.06.010
Theranostics. 2021 ;11(17): 8350-8361

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Reem Ali, Muslim Alabdullah, Mashael Algethami, Adel Alblihy, Islam Miligy, Ahmed Shoqafi, Katia A Mesquita, Tarek Abdel-Fatah, Stephen Yt Chan, Pei Wen Chiang, Nigel P Mongan, Emad A Rakha, Alan E Tomkinson, Srinivasan Madhusudan.

  Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown. Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells. Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids. Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

Keywords:  DNA repair; LIG1; LIG1 inhibitor; LIG3; LIG4; Ovarian cancer; Predictive bimarker; Prognostics; Synthetic lethality

DOI:  https://doi.org/10.7150/thno.51456
Cancer Lett. 2021 Aug 10. pii: S0304-3835(21)00395-5. [Epub ahead of print]

Inhibition of the DSB repair protein RAD51 potentiates the cytotoxic efficacy of doxorubicin via promoting apoptosis-related death pathways.

Leonie Schürmann, Lena Schumacher, Katharina Roquette, Anamaria Brozovic, Gerhard Fritz.

  The anthracycline derivative doxorubicin (Doxo) induces DNA double-strand breaks (DSBs) by inhibition of DNA topoisomerase type II. Defective mismatch repair (MMR) contributes to Doxo resistance and has been reported for colon and mammary carcinomas. Here, we investigated the outcome of pharmacological inhibition of various DNA repair-related mechanisms on Doxo-induced cytotoxicity employing MMR-deficient HCT-116 colon carcinoma cells. Out of different inhibitors tested (i.e. HDACi, PARPi, MRE11i, RAD52i, RAD51i), we identified the RAD51-inhibitor B02 as the most powerful compound to synergistically increase Doxo-induced cytotoxicity. B02-mediated synergism rests on pleiotropic mechanisms, including pronounced G2/M arrest, damage to mitochondria and caspase-driven apoptosis. Of note, B02 also promotes the cytotoxicity of oxaliplatin and 5-fluoruracil (5-FU) in HCT-116 cells and, furthermore, also increases Doxo-induced cytotoxicity in MMR-proficient colon and mammary carcinoma cells. Summarizing, pharmacological inhibition of RAD51 is suggested to synergistically increase the cytotoxic efficacy of various types of conventional anticancer drugs in different tumor entities. Hence, pre-clinical in vivo studies are preferable to determine the therapeutic window of B02 in a clinically oriented therapeutic regimen.

Keywords:  DNA damage Response; DNA repair; Doxorubicin; RAD51 inhibition

DOI:  https://doi.org/10.1016/j.canlet.2021.08.006
Nucleic Acids Res. 2021 Aug 11. pii: gkab685. [Epub ahead of print]

Cancer cells are highly susceptible to accumulation of templated insertions linked to MMBIR.

Beth Osia, Thamer Alsulaiman, Tyler Jackson, Juraj Kramara, Suely Oliveira, Anna Malkova.

Microhomology-mediated break-induced replication (MMBIR) is a DNA repair pathway initiated by polymerase template switching at microhomology, which can produce templated insertions that initiate chromosomal rearrangements leading to neurological and metabolic diseases, and promote complex genomic rearrangements (CGRs) found in cancer. Yet, how often templated insertions accumulate from processes like MMBIR in genomes is poorly understood due to difficulty in directly identifying these events by whole genome sequencing (WGS). Here, by using our newly developed MMBSearch software, we directly detect such templated insertions (MMB-TIs) in human genomes and report substantial differences in frequency and complexity of MMB-TI events between normal and cancer cells. Through analysis of 71 cancer genomes from The Cancer Genome Atlas (TCGA), we observed that MMB-TIs readily accumulate de novo across several cancer types, with particularly high accumulation in some breast and lung cancers. By contrast, MMB-TIs appear only as germline variants in normal human fibroblast cells, and do not accumulate as de novo somatic mutations. Finally, we performed WGS on a lung adenocarcinoma patient case and confirmed MMB-TI-initiated chromosome fusions that disrupted potential tumor suppressors and induced chromothripsis-like CGRs. Based on our findings we propose that MMB-TIs represent a trigger for widespread genomic instability and tumor evolution.

DOI: https://doi.org/10.1093/nar/gkab685
Cancer Discov. 2020 Oct;10(10): 1438

Relapse-Driving AML Cells Exhibit Pyrimidine-Synthesis Dependency.

Residual acute myeloid leukemia (AML) cells required bone marrow stromal cell-derived aspartate.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2020-118
Trends Cell Biol. 2021 Aug 09. pii: S0962-8924(21)00145-8. [Epub ahead of print]

DNA replication: the recombination connection.

Esther A Epum, James E Haber.

  Failure to complete DNA replication is one of the major sources of genome instability leading to aneuploidy, chromosome breakage, and chromosome rearrangements that are associated with human cancer. One of the surprising revelations of the past decade is that the completion of replication at so-called common fragile sites (CFS) occurs very late in the cell cycle - at mitosis - through a process termed MiDAS (mitotic DNA synthesis). MiDAS is strongly related to another cancer-promoting phenomenon: the activation of alternative lengthening of telomeres (ALT). Our understanding of the mechanisms of ALT and MiDAS in mammalian cells has drawn heavily from recent advances in the study of break-induced replication (BIR), especially in budding yeast. We provide new insights into the BIR, MiDAS, and ALT pathways and their shared similarities.

Keywords:  alternative lengthening of telomeres (ALT); break-induced replication (BIR); homologous recombination; mitotic DNA synthesis (MiDAS)

DOI:  https://doi.org/10.1016/j.tcb.2021.07.005
Sci Adv. 2021 Aug;pii: eabf3641. [Epub ahead of print]7(33):

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.

Nicholas A W Bell, Philip J Haynes, Katharina Brunner, Taiana Maia de Oliveira, Maria M Flocco, Bart W Hoogenboom, Justin E Molloy.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

DOI: https://doi.org/10.1126/sciadv.abf3641
Nucleic Acids Res. 2021 Aug 13. pii: gkab692. [Epub ahead of print]

A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family.

Hannah T Baddock, Joseph A Newman, Yuliana Yosaatmadja, Marcin Bielinski, Christopher J Schofield, Opher Gileadi, Peter J McHugh.

The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

DOI: https://doi.org/10.1093/nar/gkab692
Biochem Biophys Res Commun. 2021 Aug 04. pii: S0006-291X(21)01139-6. [Epub ahead of print]572 191-196

Mechanism of chromosome rearrangement arising from single-strand breaks.

Palina Kot, Takaaki Yasuhara, Atsushi Shibata, Miyako Hirakawa, Yu Abe, Motohiro Yamauchi, Naoki Matsuda.

  Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.

Keywords:  Chromosome rearrangement; Double-strand breaks; Homologous recombination; Replication; Single-strand breaks

DOI:  https://doi.org/10.1016/j.bbrc.2021.08.001
Cancer Discov. 2020 Aug;10(8): OF7

Histone Methylation Connects DNA Repair to Metabolism in Cancer Cells.

Metabolites produced in cancer cells interfered with resolution of DNA double-strand breaks.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2020-089
Plant Cell. 2021 Aug 10. pii: koab204. [Epub ahead of print]

Different DNA repair pathways are involved in single-strand break-induced genomic changes in plants.

Felix Wolter, Patrick Schindele, Natalja Beying, Armin Scheben, Holger Puchta.

In nature, single-strand breaks (SSBs) in DNA occur more frequently (by orders of magnitude) than double-strand breaks (DSBs). SSBs induced by the CRISPR/Cas9 nickase at a distance of 50 to 100 bp on opposite strands are highly mutagenic, leading to insertions/deletions (InDels), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to be important for genome evolution. We investigated the distance at which paired 5' overhanging SSBs are mutagenic and which DNA repair pathways are essential for insertion formation in Arabidopsis thaliana. We were able to detect InDel formation up to a distance of 250 bp, although with much reduced efficiency. Surprisingly, the loss of the classical non-homologous end joining (NHEJ) pathway factors KU70 or DNA ligase 4 (LIG4) completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair as templated insertions from ectopic sites. As SSBs can also be repaired using homology, we furthermore asked whether the classical homologous recombination pathway is involved in this process in plants. The fact that RAD54 is not required for homology-mediated SSB repair demonstrates that the mechanisms for DSB- and SSB-induced homologous recombination differ in plants.

DOI: https://doi.org/10.1093/plcell/koab204
Mol Biol Cell. 2021 Aug 11. mbcE20100680

Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage.

Verena Hurst, Kiran Challa, Kenji Shimada, Susan M Gasser.

Upon induction of DNA damage with 405 nm laser light, proteins involved in Base Excision Repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by Latrunculin B, Cytochalasin B or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by Nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin, but also of tubulin. Our study is one of few linking cytoskeletal integrity to BER. [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E20-10-0680
Curr Opin Genet Dev. 2021 Aug 04. pii: S0959-437X(21)00094-0. [Epub ahead of print]71 129-135

Recombination-dependent replication: new perspectives from site-specific fork barriers.

Antony Carr, Sarah Lambert.

Replication stress (RS) is intrinsic to normal cell growth, is enhanced by exogenous factors and elevated in many cancer cells due to oncogene expression. Most genetic changes are a result of RS and the mechanisms by which cells tolerate RS has received considerable attention because of the link to cancer evolution and opportunities for cancer cell-specific therapeutic intervention. Site-specific replication fork barriers have provided unique insights into how cells respond to RS and their recent use has allowed a deeper understanding of the mechanistic and spatial mechanism that restart arrested forks and how these correlate with RS-dependent mutagenesis. Here we review recent data from site-specific fork arrest systems used in yeast and highlight their strengths and limitations.

DOI: https://doi.org/10.1016/j.gde.2021.07.008
Elife. 2021 Aug 12. pii: e62592. [Epub ahead of print]10

A feedback loop between the androgen receptor and 6-phosphogluoconate dehydrogenase (6PGD) drives prostate cancer growth.

Joanna L Gillis, Josephine A Hinneh, Natalie K Ryan, Swati Irani, Max Moldovan, Lake-Ee Quek, Raj K Shrestha, Adrienne R Hanson, Jianling Xie, Andrew J Hoy, Jeff Holst, Margaret M Centenera, Ian G Mills, David J Lynn, Luke A Selth, Lisa M Butler.

  Alterations to the androgen receptor (AR) signalling axis and cellular metabolism are hallmarks of prostate cancer. This study provides insight into both hallmarks by uncovering a novel link between AR and the pentose phosphate pathway (PPP). Specifically, we identify 6-phosphogluoconate dehydrogenase (6PGD) as an androgen-regulated gene that is upregulated in prostate cancer. AR increased the expression of 6PGD indirectly via activation of sterol regulatory element binding protein 1 (SREBP1). Accordingly, loss of 6PGD, AR or SREBP1 resulted in suppression of PPP activity, as revealed by 1,2-13C2 glucose metabolic flux analysis. Knockdown of 6PGD also impaired growth and elicited death of prostate cancer cells, at least in part due to increased oxidative stress. We investigated the therapeutic potential of targeting 6PGD using two specific inhibitors, physcion and S3, and observed substantial anti-cancer activity in multiple models of prostate cancer, including aggressive, therapy-resistant models of castration-resistant disease as well as prospectively-collected patient-derived tumour explants. Targeting of 6PGD was associated with two important tumour-suppressive mechanisms: first, increased activity of the AMP-activated protein kinase (AMPK), which repressed anabolic growth-promoting pathways regulated by ACC1 and mTOR; and second, enhanced AR ubiquitylation, associated with a reduction in AR protein levels and activity. Supporting the biological relevance of positive feedback between AR and PGD, pharmacological co-targeting of both factors was more effective in suppressing the growth of prostate cancer cells than single agent therapies. Collectively, this work provides new insight into the dysregulated metabolism of prostate cancer and provides impetus for further investigation of co-targeting AR and the PPP as a novel therapeutic strategy.

Keywords:  cancer biology; human

DOI:  https://doi.org/10.7554/eLife.62592