bims-numges 2021-11-21 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–11–21
forty papers selected by
Sean Rudd, Karolinska Institutet

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia.
APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.
Visualizing replication fork encounters with DNA interstrand crosslinks.
A method to sequence genomic sites of mitotic DNA synthesis in mammalian cells.
Methionine synthase supports tumour tetrahydrofolate pools.
Role of human nucleoside transporters in pancreatic cancer and chemoresistance.
HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications.
Nucleolar GTPase Bms1 displaces Ttf1 from RFB-sites to balance progression of rDNA transcription and replication.
RNF19A-mediated ubiquitination of BARD1 prevents BRCA1/BARD1-dependent homologous recombination.
Genomic mapping of DNA-repair reaction intermediates in living cells with engineered DNA structure-trap proteins.
Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA.
Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER).
Synthesis and polymerase bypass studies of DNA-peptide and DNA-protein conjugates.
Characterization of DNA-PK-bound end fragments using GLASS-ChIP.
Putative role of uncoupling proteins in mitochondria-nucleus communications and DNA damage response.
Single-molecule imaging of replication fork conflicts at genomic DNA G4 structures in human cells.
Monitoring the replication of structured DNA through heritable epigenetic change.
Untangling the clinicopathological significance of MRE11-RAD50-NBS1 complex in sporadic breast cancers.
MicroRNAs and the DNA damage response: How is cell fate determined?
Comprehensive DNA repair gene expression analysis and its prognostic significance in acute myeloid leukemia.
Light activation and deactivation of Cas9 for DNA repair studies.
The ubiquitin E3 ligase FBXO22 degrades PD-L1 and sensitizes cancer cells to DNA damage.
Methionine synthase is essential for cancer cell proliferation in physiological folate environments.
Eukaryotic DNA replication with purified budding yeast proteins.
Genome-wide mapping of G-quadruplex structures with CUT&Tag.
Parp1 promotes sleep, which enhances DNA repair in neurons.
Analysis of repair of replication-born double-strand breaks by sister chromatid recombination in yeast.
PD-L1 sustains chronic, cancer cell-intrinsic responses to type I interferon, enhancing resistance to DNA damage.
A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.
Histone N-terminal acetyltransferase NAA40 links one-carbon metabolism to chemoresistance.
ATRX proximal protein associations boast roles beyond histone deposition.
Zipper head mechanism of telomere synthesis by human telomerase.
Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.
The Pentose Phosphate Pathway in Cancer: Regulation and Therapeutic Opportunities.
PARP Inhibitors - Trapped in a Toxic Love Affair.
R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.
NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix.
piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair.
Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27.
Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells.

Nat Cancer. 2021 Oct;2(10): 1002-1017

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia.

Melissa B Pappalardi, Kathryn Keenan, Mark Cockerill, Wendy A Kellner, Alexandra Stowell, Christian Sherk, Kristen Wong, Sarath Pathuri, Jacques Briand, Michael Steidel, Philip Chapman, Arthur Groy, Ashley K Wiseman, Charles F McHugh, Nino Campobasso, Alan P Graves, Emma Fairweather, Thilo Werner, Ali Raoof, Roger J Butlin, Lourdes Rueda, John R Horton, David T Fosbenner, Cunyu Zhang, Jessica L Handler, Morris Muliaditan, Makda Mebrahtu, Jon-Paul Jaworski, Dean E McNulty, Charlotte Burt, H Christian Eberl, Amy N Taylor, Thau Ho, Susan Merrihew, Shawn W Foley, Anna Rutkowska, Mei Li, Stuart P Romeril, Kristin Goldberg, Xing Zhang, Christopher S Kershaw, Marcus Bantscheff, Anthony J Jurewicz, Elisabeth Minthorn, Paola Grandi, Mehul Patel, Andrew B Benowitz, Helai P Mohammad, Aidan G Gilmartin, Rab K Prinjha, Donald Ogilvie, Christopher Carpenter, Dirk Heerding, Stephen B Baylin, Peter A Jones, Xiaodong Cheng, Bryan W King, Juan I Luengo, Allan M Jordan, Ian Waddell, Ryan G Kruger, Michael T McCabe.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.
Front Cell Dev Biol. 2021 ;9 738502

APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.

Md Akram Hossain, Yunfeng Lin, Garrett Driscoll, Jia Li, Anne McMahon, Joshua Matos, Haichao Zhao, Daisuke Tsuchimoto, Yusaku Nakabeppu, Jianjun Zhao, Shan Yan.

  The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3'-5' exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

Keywords:  APE2; ATR-Chk1; DNA damage response; DNA double-strand breaks; DNA single- strand breaks; genome integrity

DOI:  https://doi.org/10.3389/fcell.2021.738502
Methods Enzymol. 2021 ;pii: S0076-6879(21)00358-X. [Epub ahead of print]661 53-75

Visualizing replication fork encounters with DNA interstrand crosslinks.

Ryan C James, Marina A Bellani, Jing Zhang, Jing Huang, Althaf Shaik, Durga Pokharel, Himabindu Gali, Julia Gichimu, Arun K Thazhathveetil, Michael M Seidman.

  Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.

Keywords:  DNA fiber; DONSON; FANCM; Interstrand crosslink; Replication stress; Replication traverse

DOI:  https://doi.org/10.1016/bs.mie.2021.08.015
Methods Enzymol. 2021 ;pii: S0076-6879(21)00356-6. [Epub ahead of print]661 283-304

A method to sequence genomic sites of mitotic DNA synthesis in mammalian cells.

Jonathan Mailler, Laura Padayachy, Thanos D Halazonetis.

  Under normal conditions, the genome of eukaryotic cells is faithfully replicated during S phase. However, in cells exposed to DNA polymerase inhibitors, some regions of the genome may fail to be replicated prior to mitotic entry. To prevent chromosomal breakage and loss of genomic information, mitotic DNA synthesis (MiDAS) completes replication of the genome prior to the onset of anaphase. We have developed a protocol that allows one to map the genomic regions that are replicated by MiDAS in mammalian cells. The protocol involves incorporation of a thymidine analog in nascent DNA in mitotic cells and then capture and high throughput sequencing of the nascent DNA. With this approach, sites of MiDAS can be identified at high resolution.

Keywords:  Common fragile sites; DNA replication; DNA replication stress; MIDAS; Mitotic DNA synthesis

DOI:  https://doi.org/10.1016/bs.mie.2021.08.013
Nat Metab. 2021 Nov;3(11): 1512-1520

Methionine synthase supports tumour tetrahydrofolate pools.

Jonathan M Ghergurovich, Xincheng Xu, Joshua Z Wang, Lifeng Yang, Rolf-Peter Ryseck, Lin Wang, Joshua D Rabinowitz.

Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF), which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using isotope tracing that MTR is only a minor source of methionine in cell culture, tissues or xenografted tumours. Instead, MTR is required for cells to avoid folate trapping and assimilate 5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumour cells leads to folate trapping, purine synthesis stalling, nucleotide depletion and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role in liberating THF for use in one-carbon metabolism.

DOI: https://doi.org/10.1038/s42255-021-00465-w
World J Gastroenterol. 2021 Oct 28. 27(40): 6844-6860

Role of human nucleoside transporters in pancreatic cancer and chemoresistance.

Carly Jade Carter, Ahmed H Mekkawy, David L Morris.

  The prognosis of pancreatic cancer is poor with the overall 5-year survival rate of less than 5% changing minimally over the past decades and future projections predicting it developing into the second leading cause of cancer related mortality within the next decade. Investigations into the mechanisms of pancreatic cancer development, progression and acquired chemoresistance have been constant for the past few decades, thus resulting in the identification of human nucleoside transporters and factors affecting cytotoxic uptake via said transporters. This review summaries the aberrant expression and role of human nucleoside transports in pancreatic cancer, more specifically human equilibrative nucleoside transporter 1/2 (hENT1, hENT2), and human concentrative nucleoside transporter 1/3 (hCNT1, hCNT3), while briefly discussing the connection and importance between these nucleoside transporters and mucins that have also been identified as being aberrantly expressed in pancreatic cancer. The review also discusses the incidence, current diagnostic techniques as well as the current therapeutic treatments for pancreatic cancer. Furthermore, we address the importance of chemoresistance in nucleoside analogue drugs, in particular, gemcitabine and we discuss prospective therapeutic treatments and strategies for overcoming acquired chemoresistance in pancreatic cancer by the enhancement of human nucleoside transporters as well as the potential targeting of mucins using a combination of mucolytic compounds with cytotoxic agents.

Keywords:  Gemcitabine; Human concentrative nucleotide transporters; Human equilibrative nucleoside transporters; Human nucleoside transporters; Mucins; Pancreatic cancer

DOI:  https://doi.org/10.3748/wjg.v27.i40.6844
Nat Commun. 2021 Nov 18. 12(1): 6675

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications.

Marie-France Langelier, Ramya Billur, Aleksandr Sverzhinsky, Ben E Black, John M Pascal.

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This "hit and run" mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

DOI: https://doi.org/10.1038/s41467-021-27043-8
J Mol Cell Biol. 2021 Nov 13. pii: mjab074. [Epub ahead of print]

Nucleolar GTPase Bms1 displaces Ttf1 from RFB-sites to balance progression of rDNA transcription and replication.

Yanqing Zhu, Yong Wang, Boxiang Tao, Jinhua Han, Hong Chen, Qinfang Zhu, Ling Huang, Yinan He, Jian Hong, Yunqin Li, Jun Chen, Jun Huang, Li Jan Lo, Jinrong Peng.

  18S, 5.8S, and 28S ribosomal RNAs (rRNAs) are co-transcribed as a pre-ribosomal RNA (pre-rRNA) from the rDNA by RNA polymerase I whose activity is vigorous during the S-phase, leading to a conflict with rDNA replication. This conflict is resolved partly by replication-fork-barrier sequences (RFB-sites) located downstream of the rDNA and RFB-binding proteins such as Ttf1. However, how Ttf1 is displaced from RFB-sites to allow replication-fork progression remains elusive. Here, we reported that loss-of-function of Bms1l, a nucleolar GTPase, upregulates rDNA transcription, causes replication-fork stall, and arrests cell cycle at the S-to-G2 transition; however, the G1-to-S transition is constitutively active characterized by persisting DNA synthesis. Concomitantly, ubf, tif-IA, and taf1b marking rDNA transcription, Chk2, Rad51, and p53 marking DNA-damage response, and Rpa2, PCNA, Fen1, and Ttf1 marking replication-fork stall are all highly elevated in bms1l mutants. We found that Bms1 interacts with Ttf1 in addition to Rc1l. Finally, we identified RFB-sites for zebrafish Ttf1 through chromatin immunoprecipitation sequencing and showed that Bms1 disassociates the Ttf1‒RFB complex with its GTPase activity. We propose that Bms1 functions to balance rDNA transcription and replication at the S-phase through interaction with Rcl1 and Ttf1, respectively. TTF1 and Bms1 together might impose an S-phase checkpoint at the rDNA loci.

Keywords:  Bms1; GTPase; Ttf1; cell cycle; nucleolus; rDNA; replication-fork barrier; ribosome small subunit processome; zebrafish

DOI:  https://doi.org/10.1093/jmcb/mjab074
Nat Commun. 2021 Nov 17. 12(1): 6653

RNF19A-mediated ubiquitination of BARD1 prevents BRCA1/BARD1-dependent homologous recombination.

Qian Zhu, Jinzhou Huang, Hongyang Huang, Huan Li, Peiqiang Yi, Jake A Kloeber, Jian Yuan, Yuping Chen, Min Deng, Kuntian Luo, Ming Gao, Guijie Guo, Xinyi Tu, Ping Yin, Yong Zhang, Jun Su, Jiayi Chen, Zhenkun Lou.

BRCA1-BARD1 heterodimers act in multiple steps during homologous recombination (HR) to ensure the prompt repair of DNA double strand breaks. Dysfunction of the BRCA1 pathway enhances the therapeutic efficiency of poly-(ADP-ribose) polymerase inhibitors (PARPi) in cancers, but the molecular mechanisms underlying this sensitization to PARPi are not fully understood. Here, we show that cancer cell sensitivity to PARPi is promoted by the ring between ring fingers (RBR) protein RNF19A. We demonstrate that RNF19A suppresses HR by ubiquitinating BARD1, which leads to dissociation of BRCA1-BARD1 complex and exposure of a nuclear export sequence in BARD1 that is otherwise masked by BRCA1, resulting in the export of BARD1 to the cytoplasm. We provide evidence that high RNF19A expression in breast cancer compromises HR and increases sensitivity to PARPi. We propose that RNF19A modulates the cancer cell response to PARPi by negatively regulating the BRCA1-BARD1 complex and inhibiting HR-mediated DNA repair.

DOI: https://doi.org/10.1038/s41467-021-27048-3
Methods Enzymol. 2021 ;pii: S0076-6879(21)00391-8. [Epub ahead of print]661 155-181

Genomic mapping of DNA-repair reaction intermediates in living cells with engineered DNA structure-trap proteins.

Jingjing Liu, Qian Mei, Sadeieh Nimer, Devon M Fitzgerald, Susan M Rosenberg.

  Diverse DNA structures occur as reaction intermediates in various DNA-damage and -repair mechanisms, most of which results from replication stress. We harness the power of proteins evolutionarily optimized to bind and "trap" specific DNA reaction-intermediate structures, to quantify the structures, and discern the mechanisms of their occurrence in cells. The engineered proteins also allow genomic mapping of sites at which specific DNA structures occur preferentially, using a structure-trapping protein and ChIP-seq- or Cut-and-Tag-like methods. Genome-wide identification of sites with recurrent DNA-damage intermediates has illuminated mechanisms implicated in genome instability, replication stress, and chromosome fragility. Here, we describe X-seq, for identifying sites of recurrent four-way DNA junctions or Holliday-junctions (HJs). X-seq uses an engineered, catalysis-defective mutant of Escherichia coli RuvC HJ-specific endonuclease, RuvCDefGFP. X-seq signal indicates sites of recombinational DNA repair or replication-fork stalling and reversal. We also describe methods for genomic mapping of 3'-single-stranded DNA ends with SsEND-seq, in E. coli. Both methods allow genomic profiling of DNA-damage and -repair intermediates, which can precede genome instability, and are expected to have many additional applications including in other cells and organisms.

Keywords:  3'-ending single-stranded DNA; DNA damage; DNA repair; DNA replication stress; Holliday junctions; Replication-fork reversal

DOI:  https://doi.org/10.1016/bs.mie.2021.09.015
J Biol Chem. 2021 Nov 12. pii: S0021-9258(21)01216-3. [Epub ahead of print] 101409

Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA.

Katie H Jameson, Christian J Rudolph, Michelle Hawkins.

  The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the 'replication fork trap' region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

Keywords:  DNA; DNA binding protein; DNA helicase; DNA replication; DNA-protein interaction; Escherichia coli (E. coli); Tus; bacteria; replication termination; termination

DOI:  https://doi.org/10.1016/j.jbc.2021.101409
Methods Enzymol. 2021 ;pii: S0076-6879(21)00380-3. [Epub ahead of print]661 139-154

Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER).

Liping Liu, Neal Sugawara, Anna Malkova, James E Haber.

  A detailed understanding of how homologous recombination proceeds at the molecular level in vivo requires the ability to detect in real time the appearance of specific intermediates of DNA repair. The most detailed analysis of double-strand break (DSB) repair in eukaryotes has come from the study of budding yeast, using an inducible site-specific HO endonuclease to initiate recombination synchronously in nearly all cells of the population. Polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) methods have been used to visualize the timing of the DSB, its resection by 5' to 3' exonucleases, the binding of the Rad51 recombinase and the pairing of the Rad51 filament with a homologous donor sequence. PCR has also been used to identify the next key step: the initiation of new DNA synthesis to extend the invading stand and copy the donor template. In break-induced replication (BIR), there appears to be a very long delay between strand invasion and this primer extension step. Here we describe an alternative method, an assay for monitoring BIR elongation rate (AMBER) based on digital droplet PCR that yields a much earlier time of initial DNA synthesis. We suggest that previous methods have failed to recover the initial long, single-stranded primer extension product that is readily detected by AMBER.

Keywords:  Break-induced replication (BIR); DNA repair; Double-strand break (DSB); Kinetics; Recombination intermediates; ddPCR

DOI:  https://doi.org/10.1016/bs.mie.2021.09.004
Methods Enzymol. 2021 ;pii: S0076-6879(21)00381-5. [Epub ahead of print]661 363-405

Synthesis and polymerase bypass studies of DNA-peptide and DNA-protein conjugates.

Suresh S Pujari, Natalia Tretyakova.

  DNA-peptide (DpCs) and DNA-protein cross-links (DPCs) are DNA lesions formed when polypeptides and nuclear proteins become covalently trapped on DNA strands. DNA-protein cross-links are of enormous size and hence pose challenges to cell survival by blocking DNA replication, transcription, and repair. However, DPCs can undergo proteolytic degradation via various pathways to give shorter polypeptide chains (DpCs). The resulting DpC lesions are efficiently bypassed by translesion synthesis (TLS) DNA polymerases like κ, η, δ, etc., although polymerase bypass efficiency as well as correct base insertion depends heavily on size, sequence context, and position of peptides in DpCs. This chapter explores various synthetic methods to generate these lesions including detailed experimental procedures for the construction of DpCs and DPCs via reductive amination and oxime ligation. Further we describe biochemical experiments to investigate the effects of these lesions on DNA polymerase activity and fidelity.

Keywords:  Bioconjugation; DNA polymerase; DNA-protein cross links; Histone; Oxime ligation; Polymerase bypass; Reductive amination; Sortase mediated ligation

DOI:  https://doi.org/10.1016/bs.mie.2021.09.005
Methods Enzymol. 2021 ;pii: S0076-6879(21)00357-8. [Epub ahead of print]661 205-217

Characterization of DNA-PK-bound end fragments using GLASS-ChIP.

Rajashree A Deshpande, Tanya T Paull.

  Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent Protein Kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.

Keywords:  DNA repair; DNA-PK; Double-strand break; MRN

DOI:  https://doi.org/10.1016/bs.mie.2021.08.014
J Biosci. 2021 ;pii: 99. [Epub ahead of print]46

Putative role of uncoupling proteins in mitochondria-nucleus communications and DNA damage response.

Yahan Niu, Chang Liu, Rui Zhang, Ying Jing, Donghai Li.

Mitochondria-nucleus communications and DNA damage response (DDR) play roles in cellular stress and closely associate with a range of diseases. Mitochondrial uncoupling proteins (UCPs) are capable of uncoupling mitochondrial oxidative phosphorylation and protecting against oxidative stress. However, the potential role of UCPs in DDR and DDR-related mitochondria-nucleus communications remains unknown. The review deduces UCPs functions in mitochondria-nucleus communications implicated in metabolite regulation (e.g., reactive oxygen species) and Ca2+ signaling, and in DDR (e.g., base excision repair, double-strand DNA break repair, mitophagy and nuclear DNA degradation). Represented are shared microRNAs that regulate UCPs and DDR. It would provide novel insight into UCPs-mediated mitochondria-nucleus communications and DDR, and potentially promote drug target identification, drug discovery and clinical therapy of DDR-related diseases.
Methods Enzymol. 2021 ;pii: S0076-6879(21)00351-7. [Epub ahead of print]661 77-94

Single-molecule imaging of replication fork conflicts at genomic DNA G4 structures in human cells.

Wei Ting C Lee, Dipika Gupta, Eli Rothenberg.

  DNA G-quadruplexes (G4s) are stable, non-canonical DNA secondary structures formed within guanine(G)-rich sequences. While extensively studied in vitro, evidence of the occurrence of G4s in vivo has only recently emerged. The formation of G4 structures may pose an obstacle for diverse DNA transactions including replication, which is linked to mutagenesis and genomic instability. A fundamental question in the field has been whether and how the formation of G4s is coupled to the progression of replication forks. This process has remained undefined largely due to the lack of experimental approaches capable of monitoring the presence of G4s and their association with the replication machinery in cells. Here, we describe a detailed multicolor single-molecule localization microscopy (SMLM) protocol for detecting nanoscale spatial-association of DNA G4s with the cellular replisome complex. This method offers a unique platform for visualizing the mechanisms of G4 formation at the molecular level, as well as addressing key biological questions as to the functional roles of these structures in the maintenance of genome integrity.

Keywords:  DNA G-quadruplex; DNA replication; Single-molecule localization microscopy; Super-resolution microscopy

DOI:  https://doi.org/10.1016/bs.mie.2021.08.008
Methods Enzymol. 2021 ;pii: S0076-6879(21)00352-9. [Epub ahead of print]661 35-51

Monitoring the replication of structured DNA through heritable epigenetic change.

Guillaume Guilbaud, Julian E Sale.

  DNA can adopt non-B form structures that create significant blocks to DNA synthesis and seeking understanding of the mechanisms cells use to resolve such impediments continues to be a very active area of research. However, the ability to monitor the stalling of DNA synthesis at specific sites in the genome in living cells, of central importance to elucidating these mechanisms, poses a significant technical challenge. Replisome stalling is often transient with only a small fraction of events leading to detectable genetic changes, making traditional reporter assays insensitive to the stalling event per se. On the other hand, the imprint stalling leaves on the epigenome can be exploited as a form of biological 'tape recorder' that captures episodes of fork stalling as heritable changes in histone modifications and in transcription. Here we describe a detailed protocol for monitoring DNA structure-dependent epigenetic instability of the BU-1 locus in the avian cell line DT40, which has proved a sensitive tool for understanding the mechanisms by which structured DNA is replicated in a vertebrate system.

Keywords:  DNA helicases; DNA polymerases; DNA replication; DNA secondary structure; G quadruplex

DOI:  https://doi.org/10.1016/bs.mie.2021.08.009
NPJ Breast Cancer. 2021 Nov 15. 7(1): 143

Untangling the clinicopathological significance of MRE11-RAD50-NBS1 complex in sporadic breast cancers.

Adel Alblihy, Ahmed Shoqafi, Michael S Toss, Mashael Algethami, Anna E Harris, Jennie N Jeyapalan, Tarek Abdel-Fatah, Juliette Servante, Stephen Y T Chan, Andrew Green, Nigel P Mongan, Emad A Rakha, Srinivasan Madhusudan.

The MRE11-RAD50-NBS1 (MRN) complex is critical for genomic stability. Although germline mutations in MRN may increase breast cancer susceptibility, such mutations are extremely rare. Here, we have conducted a comprehensive clinicopathological study of MRN in sporadic breast cancers. We have protein expression profiled for MRN and a panel of DNA repair factors involved in double-strand break repair (BRCA1, BRCA2, ATM, CHK2, ATR, Chk1, pChk1, RAD51, γH2AX, RPA1, RPA2, DNA-PKcs), RECQ DNA helicases (BLM, WRN, RECQ1, RECQL4, RECQ5), nucleotide excision repair (ERCC1) and base excision repair (SMUG1, APE1, FEN1, PARP1, XRCC1, Pol β) in 1650 clinical breast cancers. The prognostic significance of MRE11, RAD50 and NBS1 transcripts and their microRNA regulators (hsa-miR-494 and hsa-miR-99b) were evaluated in large clinical datasets. Expression of MRN components was analysed in The Cancer Genome Atlas breast cancer cohort. We show that low nuclear MRN is linked to aggressive histopathological phenotypes such as high tumour grade, high mitotic index, oestrogen receptor- and high-risk Nottingham Prognostic Index. In univariate analysis, low nuclear MRE11 and low nuclear RAD50 were associated with poor survival. In multivariate analysis, low nuclear RAD50 remained independently linked with adverse clinical outcomes. Low RAD50 transcripts were also linked with reduced survival. In contrast, overexpression of hsa-miR-494 and hsa-miR-99b microRNAs was associated with poor survival. We observed large-scale genome-wide alterations in MRN-deficient tumours contributing to aggressive behaviour. We conclude that MRN status may be a useful tool to stratify tumours for precision medicine strategies.

DOI: https://doi.org/10.1038/s41523-021-00350-5
DNA Repair (Amst). 2021 Nov 02. pii: S1568-7864(21)00201-9. [Epub ahead of print]108 103245

MicroRNAs and the DNA damage response: How is cell fate determined?

Hartwig Visser, Adam D Thomas.

  It is becoming clear that the DNA damage response orchestrates an appropriate response to a given level of DNA damage, whether that is cell cycle arrest and repair, senescence or apoptosis. It is plausible that the alternative regulation of the DNA damage response (DDR) plays a role in deciding cell fate following damage. MicroRNAs (miRNAs) are associated with the transcriptional regulation of many cellular processes. They have diverse functions, affecting, presumably, all aspects of cell biology. Many have been shown to be DNA damage inducible and it is conceivable that miRNA species play a role in deciding cell fate following DNA damage by regulating the expression and activation of key DDR proteins. From a clinical perspective, miRNAs are attractive targets to improve cancer patient outcomes to DNA-damaging chemotherapy. However, cancer tissue is known to be, or to become, well adapted to DNA damage as a means of inducing chemoresistance. This frequently results from an altered DDR, possibly owing to miRNA dysregulation. Though many studies provide an overview of miRNAs that are dysregulated within cancerous tissues, a tangible, functional association is often lacking. While miRNAs are well-documented in 'ectopic biology', the physiological significance of endogenous miRNAs in the context of the DDR requires clarification. This review discusses miRNAs of biological relevance and their role in DNA damage response by potentially 'fine-tuning' the DDR towards a particular cell fate in response to DNA damage. MiRNAs are thus potential therapeutic targets/strategies to limit chemoresistance, or improve chemotherapeutic efficacy.

Keywords:  ATM; Apoptosis; Cell fate; DNA damage response; MiRNA; P53

DOI:  https://doi.org/10.1016/j.dnarep.2021.103245
Hematology. 2021 Dec;26(1): 904-913

Comprehensive DNA repair gene expression analysis and its prognostic significance in acute myeloid leukemia.

Sholhui Park, Yi-Jun Kim, Hee Jin Huh, Hae-Sun Chung, Miae Lee, Young Mi Park, Yeung Chul Mun, Chu-Myong Seong, Jungwon Huh.

   BACKGROUND: Deficiency in DNA damage response (DDR) pathway and accumulation of DNA damage increases mutation rates resulting in genomic instability and eventually increases the risk of cancer. The aim of our study was to investigate expressions of DNA repair genes as new prognostic biomarkers in acute myeloid leukemia (AML).
METHODS: We utilized The Cancer Genome Atlas AML project (TCGA-LAML cohort, 15 acute promyelocytic leukemia (APL) and 155 non-APL AML) for the expression data of DNA repair genes. For validation, clinical samples (Ewha study group, 9 APL and 72 non-APL AML patients) were analyzed for the expression of 22 DNA repair genes using a custom RT2 Profiler PCR Array.
RESULTS: APL patients presented significantly lower expression of DNA repair genes than non-APL AML patients in both study groups. Among non-APL AML patients, high expression levels of PARP1, XRCC1, and RAD51 were associated with poor overall survival (OS) probability in both study groups. Furthermore, Cox regression analysis showed that increased expression levels of PARP1, XRCC1, RAD51, BRCA1 and MRE11A could be independent risk factors for OS in the Ewha study group. Among non-APL patients of the Ewha study group, the OS probability of DDR-overexpressed group with at least one gene or more showing Z score greater than 1.5 was poorer than that of DDR non-overexpressed group.
CONCLUSION: In the current study, the DNA repair gene expression profile of APL patients was different from that of non-APL AML patients. Overexpression of DNA repair genes could be a poor prognostic biomarker in non-APL AML.

Keywords:  DNA damage response; DNA repair gene; PARP inhibitors; TCGA; acute myeloid leukemia; biomarker; gene expression profile; prognosis

DOI:  https://doi.org/10.1080/16078454.2021.1997196
Methods Enzymol. 2021 ;pii: S0076-6879(21)00350-5. [Epub ahead of print]661 219-249

Light activation and deactivation of Cas9 for DNA repair studies.

Roger S Zou, Taekjip Ha.

  DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response: DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.

Keywords:  Biophysics; CRISPR; Cas9; DNA damage; DNA repair

DOI:  https://doi.org/10.1016/bs.mie.2021.08.007
Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2112674118. [Epub ahead of print]118(47):

The ubiquitin E3 ligase FBXO22 degrades PD-L1 and sensitizes cancer cells to DNA damage.

Sarmishtha De, Elise G Holvey-Bates, Kala Mahen, Belinda Willard, George R Stark.

  High expression of programmed death-ligand 1 (PD-L1) in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies. We find that high expression of the ubiquitin E3 ligase FBXO22 sensitizes nonsmall cell lung cancer (NSCLC) cells to ionizing radiation (IR) and cisplatin, and that activation of FBXO22 by phosphorylation is necessary for this function. Importantly, FBXO22 activates PD-L1 ubiquitination and degradation, which in turn increases the sensitivity of NSCLC cells to DNA damage. Cyclin-dependent kinase 5 (CDK5), aberrantly active in cancer cells, plays a crucial role in increasing the expression of PD-L1 in medulloblastoma [R. D. Dorand et al, Science 353, 399-403 (2016)]. We show in NSCLC cells that inhibiting CDK5 or reducing its expression increases the level of FBXO22, decreases that of PD-L1, and increases the sensitivity of the cells to DNA damage. We conclude that FBXO22 is a substrate of CDK5, and that inhibiting CDK5 reduces PD-L1 indirectly by increasing FBXO22. Pairing inhibitors of CDK5 with immune checkpoint inhibitors may increase the efficacy of immune checkpoint blockade alone or in combination with DNA-damaging therapies.

Keywords:  CDK5; FBXO22; PD-L1; lung cancer

DOI:  https://doi.org/10.1073/pnas.2112674118
Nat Metab. 2021 Nov;3(11): 1500-1511

Methionine synthase is essential for cancer cell proliferation in physiological folate environments.

Mark R Sullivan, Alicia M Darnell, Montana F Reilly, Tenzin Kunchok, Lena Joesch-Cohen, Daniel Rosenberg, Ahmed Ali, Matthew G Rees, Jennifer A Roth, Caroline A Lewis, Matthew G Vander Heiden.

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.

DOI: https://doi.org/10.1038/s42255-021-00486-5
Methods Enzymol. 2021 ;pii: S0076-6879(21)00361-X. [Epub ahead of print]661 1-33

Eukaryotic DNA replication with purified budding yeast proteins.

Viktor Posse, Erik Johansson, John F X Diffley.

  The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.

Keywords:  Chromosome duplication; DNA replication; DNA replication assay; DNA replication in vitro; Protein expression; Protein purification; Reconstituted yeast DNA replication

DOI:  https://doi.org/10.1016/bs.mie.2021.08.018
Nucleic Acids Res. 2021 Nov 18. pii: gkab1073. [Epub ahead of print]

Genome-wide mapping of G-quadruplex structures with CUT&Tag.

Jing Lyu, Rui Shao, Philip Yuk Kwong Yung, Simon J Elsässer.

Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (ESC), observing widespread G4 formation at active promoters, active and poised enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mouse ESCs. Upon differentiation to neural progenitor cells (NPC), enhancer G4s are lost. Further, performing R-loop CUT&Tag, we demonstrate the genome-wide co-occurrence of single-stranded DNA, G4s and R loops at promoters and enhancers. We confirm that G4 structures exist independent of ongoing transcription, suggesting an intricate relationship between transcription and non-canonical DNA structures.

DOI: https://doi.org/10.1093/nar/gkab1073
Mol Cell. 2021 Nov 12. pii: S1097-2765(21)00933-3. [Epub ahead of print]

Parp1 promotes sleep, which enhances DNA repair in neurons.

David Zada, Yaniv Sela, Noa Matosevich, Adir Monsonego, Tali Lerer-Goldshtein, Yuval Nir, Lior Appelbaum.

  The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.

Keywords:  DNA damage response; Ku80; NREM; Parp1; Rad52; chromosome dynamics; homeostasis; mice; sleep; zebrafish

DOI:  https://doi.org/10.1016/j.molcel.2021.10.026
Methods Enzymol. 2021 ;pii: S0076-6879(21)00353-0. [Epub ahead of print]661 121-138

Analysis of repair of replication-born double-strand breaks by sister chromatid recombination in yeast.

Belén Gómez-González, Pedro Ortega, Andrés Aguilera.

  The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

Keywords:  DSB repair; Double-strand break; Replication; Replication-born DSB; Sister chromatid exchange; Sister chromatid recombination

DOI:  https://doi.org/10.1016/bs.mie.2021.08.010
Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2112258118. [Epub ahead of print]118(47):

PD-L1 sustains chronic, cancer cell-intrinsic responses to type I interferon, enhancing resistance to DNA damage.

HyeonJoo Cheon, Elise G Holvey-Bates, Daniel J McGrail, George R Stark.

  Programmed death ligand 1 (PD-L1), an immune-checkpoint protein expressed on cancer cells, also functions independently of the immune system. We found that PD-L1 inhibits the killing of cancer cells in response to DNA damage in an immune-independent manner by suppressing their acute response to type I interferon (IFN; IFN-I). In addition, PD-L1 plays a critical role in sustaining high levels of constitutive expression in cancer cells of a subset of IFN-induced genes, the IFN-related DNA damage resistance signature (IRDS) which, paradoxically, protects cancer cells. The cyclic GMP-AMP synthase-stimulator of the IFN genes (cGAS-STING) pathway is constitutively activated in a subset of cancer cells in the presence of high levels of PD-L1, thus leading to a constitutive, low level of IFN-β expression, which in turn increases IRDS expression. The constitutive low level of IFN-β expression is critical for the survival of cancer cells addicted to self-produced IFN-β. Our study reveals immune-independent functions of PD-L1 that inhibit cytotoxic acute responses to IFN-I and promote protective IRDS expression by supporting protective chronic IFN-I responses, both of which enhance the resistance of cancer cells to DNA damage.

Keywords:  DNA damage resistance; cGAS-STING pathway; programmed death ligand 1 (PD-L1); type I interferon (IFN-I)

DOI:  https://doi.org/10.1073/pnas.2112258118
Mol Cell. 2021 Nov 12. pii: S1097-2765(21)00909-6. [Epub ahead of print]

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Christos P Zampetidis, Panagiotis Galanos, Andriani Angelopoulou, Yajie Zhu, Aikaterini Polyzou, Timokratis Karamitros, Athanassios Kotsinas, Nefeli Lagopati, Ioanna Mourkioti, Reza Mirzazadeh, Alexandros Polyzos, Silvano Garnerone, Athanasia Mizi, Eduardo G Gusmao, Konstantinos Sofiadis, Zita Gál, Dorthe H Larsen, Dafni-Eleftheria Pefani, Marco Demaria, Aristotelis Tsirigos, Nicola Crosetto, Apolinar Maya-Mendoza, Angelos Papaspyropoulos, Konstantinos Evangelou, Jiri Bartek, Argyris Papantonis, Vassilis G Gorgoulis.

  Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Keywords:  BHLHE40; DNA damage; DNA replication; Hi-C; cancer; chromatin loop; replication stress; senescence

DOI:  https://doi.org/10.1016/j.molcel.2021.10.017
Oncogene. 2021 Nov 16.

Histone N-terminal acetyltransferase NAA40 links one-carbon metabolism to chemoresistance.

Christina Demetriadou, Anastasia Raoukka, Evelina Charidemou, Constantine Mylonas, Christina Michael, Swati Parekh, Costas Koufaris, Paris Skourides, Panagiotis Papageorgis, Peter Tessarz, Antonis Kirmizis.

Aberrant function of epigenetic modifiers plays an important role not only in the progression of cancer but also the development of drug resistance. N-alpha-acetyltransferase 40 (NAA40) is a highly specific epigenetic enzyme catalyzing the transfer of an acetyl moiety at the N-terminal end of histones H4 and H2A. Recent studies have illustrated the essential oncogenic role of NAA40 in various cancer types but its role in chemoresistance remains unclear. Here, using transcriptomic followed by metabolomic analysis in colorectal cancer (CRC) cells, we demonstrate that NAA40 controls key one-carbon metabolic genes and corresponding metabolites. In particular, through its acetyltransferase activity NAA40 regulates the methionine cycle thereby affecting global histone methylation and CRC cell survival. Importantly, NAA40-mediated metabolic rewiring promotes resistance of CRC cells to antimetabolite chemotherapy in vitro and in xenograft models. Specifically, NAA40 stimulates transcription of the one-carbon metabolic gene thymidylate synthase (TYMS), whose product is targeted by 5-fluorouracil (5-FU) and accordingly in primary CRC tumours NAA40 expression associates with TYMS levels and poorer 5-FU response. Mechanistically, NAA40 activates TYMS by preventing enrichment of repressive H2A/H4S1ph at the nuclear periphery. Overall, these findings define a novel regulatory link between epigenetics and cellular metabolism mediated by NAA40, which is harnessed by cancer cells to evade chemotherapy.

DOI: https://doi.org/10.1038/s41388-021-02113-9
PLoS Genet. 2021 Nov 15. 17(11): e1009909

ATRX proximal protein associations boast roles beyond histone deposition.

William A Scott, Erum Z Dhanji, Boris J A Dyakov, Ema S Dreseris, Jonathon S Asa, Laura J Grange, Mila Mirceta, Christopher E Pearson, Grant S Stewart, Anne-Claude Gingras, Eric I Campos.

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions.

DOI: https://doi.org/10.1371/journal.pgen.1009909
Cell Res. 2021 Nov 15.

Zipper head mechanism of telomere synthesis by human telomerase.

Futang Wan, Yongbo Ding, Yuebin Zhang, Zhenfang Wu, Shaobai Li, Lin Yang, Xiangyu Yan, Pengfei Lan, Guohui Li, Jian Wu, Ming Lei.

Telomerase, a multi-subunit ribonucleoprotein complex, is a unique reverse transcriptase that catalyzes the processive addition of a repeat sequence to extend the telomere end using a short fragment of its own RNA component as the template. Despite recent structural characterizations of human and Tetrahymena telomerase, it is still a mystery how telomerase repeatedly uses its RNA template to synthesize telomeric DNA. Here, we report the cryo-EM structure of human telomerase holoenzyme bound with telomeric DNA at resolutions of 3.5 Å and 3.9 Å for the catalytic core and biogenesis module, respectively. The structure reveals that a leucine residue Leu980 in telomerase reverse transcriptase (TERT) catalytic subunit functions as a zipper head to limit the length of the short primer-template duplex in the active center. Moreover, our structural and computational analyses suggest that TERT and telomerase RNA (hTR) are organized to harbor a preformed active site that can accommodate short primer-template duplex substrates for catalysis. Furthermore, our findings unveil a double-fingers architecture in TERT that ensures nucleotide addition processivity of human telomerase. We propose that the zipper head Leu980 is a structural determinant for the sequence-based pausing signal of DNA synthesis that coincides with the RNA element-based physical template boundary. Functional analyses unveil that the non-glycine zipper head plays an essential role in both telomerase repeat addition processivity and telomere length homeostasis. In addition, we also demonstrate that this zipper head mechanism is conserved in all eukaryotic telomerases. Together, our study provides an integrated model for telomerase-mediated telomere synthesis.

DOI: https://doi.org/10.1038/s41422-021-00586-7
Nucleic Acids Res. 2021 Nov 12. pii: gkab1022. [Epub ahead of print]

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.

Jiao An, Mengdie Yin, Jiayong Yin, Sizhong Wu, Christopher P Selby, Yanyan Yang, Aziz Sancar, Guo-Liang Xu, Maoxiang Qian, Jinchuan Hu.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

DOI: https://doi.org/10.1093/nar/gkab1022
Chemotherapy. 2021 Nov 12. 1-13

The Pentose Phosphate Pathway in Cancer: Regulation and Therapeutic Opportunities.

Noorhan Ghanem, Chirine El-Baba, Khaled Araji, Riyad El-Khoury, Julnar Usta, Nadine Darwiche.

   BACKGROUND: Tumorigenesis is associated with deregulation of nutritional requirements, intermediary metabolites production, and microenvironment interactions. Unlike their normal cell counterparts, tumor cells rely on aerobic glycolysis, through the Warburg effect.
SUMMARY: The pentose phosphate pathway (PPP) is a major glucose metabolic shunt that is upregulated in cancer cells. The PPP comprises an oxidative and a nonoxidative phase and is essential for nucleotide synthesis of rapidly dividing cells. The PPP also generates nicotinamide adenine dinucleotide phosphate, which is required for reductive metabolism and to counteract oxidative stress in tumor cells. This article reviews the regulation of the PPP and discusses inhibitors that target its main pathways. Key Message: Exploiting the metabolic vulnerability of the PPP offers potential novel therapeutic opportunities and improves patients' response to cancer therapy.

Keywords:  Cancer; Metabolism; Pentose phosphate pathway

DOI:  https://doi.org/10.1159/000519784
Cancer Res. 2021 Nov 15. 81(22): 5605-5607

PARP Inhibitors - Trapped in a Toxic Love Affair.

Dragomir B Krastev, Andrew J Wicks, Christopher J Lord.

It is often the case that when an investigational cancer drug first enters clinical development, its precise mechanism of action is unclear. This was the case for PARP inhibitors (PARPi) used to treat homologous recombination-defective cancers. In 2012, nearly a decade after the first PARPi entered clinical development, work from Murai and colleagues demonstrated that clinical PARPi not only inhibit the catalytic activity of PARP1, PARylation, but also "trap" PARP1 on DNA; this latter effect being responsible for much of the tumor cell cytotoxicity caused by these drugs. We discuss how this work not only changed our understanding about how PARPi work, but also stimulated subsequent dissection of how PARP1 carries out its normal function in the absence of inhibitor.See related article by Murai and colleagues, Cancer Res 2012;72:5588-99.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-3201
Sci Adv. 2021 Nov 19. 7(47): eabj5769

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.

Ourania Chatzidoukaki, Kalliopi Stratigi, Evi Goulielmaki, George Niotis, Alexia Akalestou-Clocher, Katerina Gkirtzimanaki, Alexandros Zafeiropoulos, Janine Altmüller, Pantelis Topalis, George A Garinis.

[Figure: see text].

DOI: https://doi.org/10.1126/sciadv.abj5769
Cell Biosci. 2021 Nov 17. 11(1): 195

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix.

Bastien Proust, Martina Radić, Nikolina Škrobot Vidaček, Cécile Cottet, Stéphane Attia, Frédéric Lamarche, Lucija Ačkar, Vlatka Godinić Mikulčić, Malgorzata Tokarska-Schlattner, Helena Ćetković, Uwe Schlattner, Maja Herak Bosnar.

   BACKGROUND: NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6.
RESULTS: We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation.
CONCLUSIONS: NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Keywords:  Mitochondria; NDP kinase; NME; RCC1L; WBSCR16; nm23

DOI:  https://doi.org/10.1186/s13578-021-00707-0
Commun Biol. 2021 Nov 19. 4(1): 1312

piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair.

Basudeb Das, Neha Jain, Bibekanand Mallick.

Resistance to doxorubicin (DOX) is an obstacle to successful sarcoma treatment and a cause of tumor relapse, with the underlying molecular mechanism still unknown. PIWI-interacting RNAs (piRNAs) have been shown to enhance patient outcomes in cancers. However, there are few or no reports on piRNAs affecting chemotherapy in cancers, including fibrosarcoma. The current study aims to investigate the relationship between piR-39980 and DOX resistance and the underlying mechanisms. We reveal that piR-39980 is less expressed in DOX-resistant HT1080 (HT1080/DOX) fibrosarcoma cells. Our results show that inhibition of piR-39980 in parental HT1080 cells induces DOX resistance by attenuating intracellular DOX accumulation, DOX-induced apoptosis, and anti-proliferative effects. Its overexpression in HT1080/DOX cells, on the other hand, increases DOX sensitivity by promoting intracellular DOX accumulation, DNA damage, and apoptosis. The dual-luciferase reporter assay indicates that piR-39980 negatively regulates RRM2 and CYP1A2 via direct binding to their 3'UTRs. Furthermore, overexpressing RRM2 induces DOX resistance of HT1080 cells by rescuing DOX-induced DNA damage by promoting DNA repair, whereas CYP1A2 confers resistance by decreasing intracellular DOX accumulation, which piR-39980 restores. This study reveals that piR-39980 could reduce fibrosarcoma resistance to DOX by modulating RRM2 and CYP1A2, implying that piRNA can be used in combination with DOX.

DOI: https://doi.org/10.1038/s42003-021-02844-1
Open Biol. 2021 Nov;11(11): 210125

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27.

Betheney R Pennycook, Alexis R Barr.

  The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.

Keywords:  cancer biology; cell cycle; cyclin-dependent kinases; proliferation

DOI:  https://doi.org/10.1098/rsob.210125
Mol Cancer Res. 2021 Nov 15. pii: molcanres.MCR-21-0366-E.2021. [Epub ahead of print]

Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells.

Liang Zhou, Xinyan Pei, Yu Zhang, Yanxia Ning, Lin Li, Xiaoyan Hu, Sri Lakshmi Chalasani, Kanika Sharma, Jewel Nkwocha, Jonathan Yu, Dipankar Bandyopadhyay, Said M Sebti, Steven Grant.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma (MM) cells. Gene expression profiling of U266 cells exposed to low (nM) Chk1 inhibitor (PF-477736) concentrations revealed STAT3 pathway-related gene down-regulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by ChIP assay, and down-regulation of STAT-3-dependent proteins. Similar findings were obtained in other MM cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. SPR analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or shRNA knockdown cells also displayed STAT3 inactivation and STAT-3-dependent protein down-regulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and down-regulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (rH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL-6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ MM cells and tumors extracted from an NSG MM xenograft model while inhibiting tumor growth. Implications: These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0366