bims-numges 2021-05-09 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒05‒09
38 papers selected by
Sean Rudd
Karolinska Institutet

Induction of APOBEC3 exacerbates DNA replication stress and chromosomal instability in early breast and lung cancer evolution.
A subset of PARP inhibitors induces lethal telomere fusion in ALT-dependent tumor cells.
SMARCAD1-mediated active replication fork stability maintains genome integrity.
Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.
Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion.
Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling.
Autism-associated vigilin depletion impairs DNA damage repair.
The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair.
NSMF promotes the replication stress-induced DNA damage response for genome maintenance.
Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461.
PARP1: Structural insights and pharmacological targets for inhibition.
The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity.
Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.
Sweet Melody or Jazz? Transcription Around DNA Double-Strand Breaks.
USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors.
A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1.
Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.
Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4.
Post-translational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.
AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.
The Contribution of Lysosomes to DNA Replication.
Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells.
A role for VCP/p97 in the processing of drug-stabilised TOP2-DNA covalent complexes.
Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase.
PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability.
Mutations in the PCNA DNA Polymerase Clamp of Saccharomyces cerevisiae Reveal Complexities of the Cell Cycle and Ploidy on Heterochromatin Assembly.
Unfolding New Roles for Guanine-Based Purines and Their Metabolizing Enzymes in Cancer and Aging Disorders.
TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.
Unresolved Recombination Intermediates Cause a RAD9-Dependent Cell Cycle Arrest in Saccharomyces cerevisiae.
Tolerance of DNA Replication Stress Is Promoted by Fumarate Through Modulation of Histone Demethylation and Enhancement of Replicative Intermediate Processing in Saccharomyces cerevisiae.
DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion.
Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches.
Laser Micro-Irradiation to Study DNA Recruitment During S Phase.
Reduced purine biosynthesis in humans after their divergence from Neandertals.
REV7: Jack of many trades.
Combined inhibition of Rad51 and Wee1 enhances cell killing in HNSCC through induction of apoptosis associated with excessive DNA damage and replication stress.
Disabling the Fanconi Anemia Pathway in Stem Cells Leads to Radioresistance and Genomic Instability.
Loss of BubR1 acetylation provokes replication stress and leads to complex chromosomal rearrangements.

Cancer Discov. 2021 May 04. pii: candisc.0725.2020. [Epub ahead of print]

Induction of APOBEC3 exacerbates DNA replication stress and chromosomal instability in early breast and lung cancer evolution.

Subramanian Venkatesan, Mihaela Angelova, Clare Puttick, Haoran Zhai, Deborah R Caswell, Wei-Ting Lu, Michelle Dietzen, Panagiotis Galanos, Konstantinos Evangelou, Roberto Bellelli, Emilia L Lim, Thomas B K Watkins, Andrew Rowan, Vitor H Teixeira, Yue Zhao, Haiquan Chen, Bryan Ngo, Lykourgos-Panagiotis Zalmas, Maise Al Bakir, Sebastijan Hobor, Eva Gronroos, Adam Pennycuick, Ersilia Nigro, Brittany B Campbell, William L Brown, Ayse U Akarca, Teresa Marafioti, Mary Y Wu, Michael Howell, Simon J Boulton, Cosetta Bertoli, Tim R Fenton, Robertus A M de Bruin, Apolinar Maya-Mendoza, Eric Santoni-Rugiu, Robert E Hynds, Vassilis G Gorgoulis, Mariam Jamal-Hanjani, Nicholas McGranahan, Reuben S Harris, Sam M Janes, Jirina Bartkova, Samuel F Bakhoum, Jiri Bartek, Nnennaya Kanu, Charles Swanton, TRACERx Consortium.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast DCIS, and in pre-invasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx pre-invasive to invasive NSCLC lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models, revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in pre-invasive disease, providing fuel for selection early in cancer evolution.

DOI: https://doi.org/10.1158/2159-8290.CD-20-0725
Sci Transl Med. 2021 May 05. pii: eabc7211. [Epub ahead of print]13(592):

A subset of PARP inhibitors induces lethal telomere fusion in ALT-dependent tumor cells.

Joydeep Mukherjee, Ajay Pandita, Chatla Kamalakar, Tor-Christian Johannessen, Shigeo Ohba, Yongjian Tang, Cecilia L Dalle-Ore, Rolf Bjerkvig, Russell O Pieper.

About 10% of all tumors, including most lower-grade astrocytoma, rely on the alternative lengthening of telomere (ALT) mechanism to resolve telomeric shortening and avoid limitations on their growth. Here, we found that dependence on the ALT mechanism made cells hypersensitive to a subset of poly(ADP-ribose) polymerase inhibitors (PARPi). We found that this hypersensitivity was not associated with PARPi-created genomic DNA damage as in most PARPi-sensitive populations but rather with PARPi-induced telomere fusion. Mechanistically, we determined that PARP1 was recruited to the telomeres of ALT-dependent cells as part of a DNA damage response. By recruiting MRE11 and BRCC3 to stabilize TRF2 at the ends of telomeres, PARP1 blocked chromosomal fusion. Exposure of ALT-dependent tumor cells to a subset of PARPi induced a conformational change in PARP1 that limited binding to MRE11 and BRCC3 and delayed release of the TRF2-mediated block on lethal telomeric fusion. These results therefore provide a basis for PARPi treatment of ALT-dependent tumors, as well as establish chromosome fusion as a biomarker of their activity.

DOI: https://doi.org/10.1126/scitranslmed.abc7211
Sci Adv. 2021 May;pii: eabe7804. [Epub ahead of print]7(19):

SMARCAD1-mediated active replication fork stability maintains genome integrity.

Calvin Shun Yu Lo, Marvin van Toorn, Vincent Gaggioli, Mariana Paes Dias, Yifan Zhu, Eleni Maria Manolika, Wei Zhao, Marit van der Does, Chirantani Mukherjee, João G S C Souto Gonçalves, Martin E van Royen, Pim J French, Jeroen Demmers, Ihor Smal, Hannes Lans, David Wheeler, Jos Jonkers, Arnab Ray Chaudhuri, Jurgen A Marteijn, Nitika Taneja.

The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

DOI: https://doi.org/10.1126/sciadv.abe7804
Mol Cell. 2021 Apr 22. pii: S1097-2765(21)00311-7. [Epub ahead of print]

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

Grazia Pellicanò, Mohammed Al Mamun, Dolores Jurado-Santiago, Sara Villa-Hernández, Xingyu Yin, Michele Giannattasio, Michael C Lanz, Marcus B Smolka, Joseph Yeeles, Katsuhiko Shirahige, Miguel García-Díaz, Rodrigo Bermejo.

  DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Keywords:  DNA polymerase ε; DNA replication checkpoint; Exo1 exonuclease; Rad53(CHK1) kinase; genome integrity maintenance; nascent strand resection; polymerase-exonuclease partitioning; replication fork stabilization; replication stress; replisome phosphorylation

DOI:  https://doi.org/10.1016/j.molcel.2021.04.006
PLoS One. 2021 ;16(5): e0251188

Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion.

Wei-Chung Tsao, Raquel Buj, Katherine M Aird, Julia M Sidorova, Kristin A Eckert.

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS G12V overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS G12V overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol α catalytic subunit (POLA1), Pol δ catalytic and p68 subunits (POLD1 and POLD3), Pol η, and Pol κ. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol η (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol α expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-RasG12V-induced polymerase depletion, as compared to Pol η-deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.

DOI: https://doi.org/10.1371/journal.pone.0251188
Nat Commun. 2021 05 05. 12(1): 2525

Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling.

Wei Ting C Lee, Yandong Yin, Michael J Morten, Peter Tonzi, Pam Pam Gwo, Diana C Odermatt, Mauro Modesti, Sharon B Cantor, Kerstin Gari, Tony T Huang, Eli Rothenberg.

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.

DOI: https://doi.org/10.1038/s41467-021-22830-9
Mol Cell Biol. 2021 May 03. pii: MCB.00082-21. [Epub ahead of print]

Autism-associated vigilin depletion impairs DNA damage repair.

Shahid Banday, Raj K Pandita, Arjamand Mushtaq, Albino Bacolla, Ulfat Syed Mir, Dharmendra Kumar Singh, Sadaf Jan, Krishna P Bhat, Clayton R Hunt, Ganesh Rao, Vijay K Charaka, John A Tainer, Tej K Pandita, Mohammad Altaf.

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, mRNA stability and is associated with autism-spectrum disorders and cancer, vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K type homology (KH) domains. Here we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX, elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 foci formation, DNA end resection and double strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU) induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer related disorders.

DOI: https://doi.org/10.1128/MCB.00082-21
Genetics. 2019 Nov 01. 213(3): 835-847

The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair.

Ece Kocak, Sarah Dykstra, Alexandra Nemeth, Catherine G Coughlin, Kasey Rodgers, Mitch McVey.

  PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.

Keywords:  DNA damage; break-induced replication; homologous recombination; polymerase; replication fork collapse

DOI:  https://doi.org/10.1534/genetics.119.302665
Nucleic Acids Res. 2021 May 08. pii: gkab311. [Epub ahead of print]

NSMF promotes the replication stress-induced DNA damage response for genome maintenance.

Min Kyung Ju, Kyeong Jin Shin, Joo Rak Lee, Keon Woo Khim, Eun A Lee, Jae Sun Ra, Byung-Gyu Kim, Han-Seul Jo, Jong Hyuk Yoon, Tae Moon Kim, Kyungjae Myung, Jang Hyun Choi, Hongtae Kim, Young Chan Chae.

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.

DOI: https://doi.org/10.1093/nar/gkab311
Sci Rep. 2021 May 07. 11(1): 9812

Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461.

Tehmina Masud, Charles Soong, Hong Xu, Justina Biele, Saelin Bjornson, Steven McKinney, Samuel Aparicio.

CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.

DOI: https://doi.org/10.1038/s41598-021-88988-w
DNA Repair (Amst). 2021 Apr 14. pii: S1568-7864(21)00081-1. [Epub ahead of print]103 103125

PARP1: Structural insights and pharmacological targets for inhibition.

Jacob O Spiegel, Bennett Van Houten, Jacob D Durrant.

  Poly(ADP-ribose) polymerase 1 (PARP1, also known as ADPRT1) is a multifunctional human ADP-ribosyltransferase. It plays a role in multiple DNA repair pathways, including the base excision repair (BER), non-homologous end joining (NHEJ), homologous recombination (HR), and Okazaki-fragment processing pathways. In response to DNA strand breaks, PARP1 covalently attaches ADP-ribose moieties to arginine, glutamate, aspartate, cysteine, lysine, and serine acceptor sites on both itself and other proteins. This signal recruits DNA repair proteins to the site of DNA damage. PARP1 binding to these sites enhances ADP-ribosylation via allosteric communication between the distant DNA binding and catalytic domains. In this review, we provide a general overview of PARP1 and emphasize novel potential approaches for pharmacological inhibition. Clinical PARP1 inhibitors bind the catalytic pocket, where they directly interfere with ADP-ribosylation. Some inhibitors may further enhance potency by "trapping" PARP1 on DNA via an allosteric mechanism, though this proposed mode of action remains controversial. PARP1 inhibitors are used clinically to treat some cancers, but resistance is common, so novel pharmacological approaches are urgently needed. One approach may be to design novel small molecules that bind at inter-domain interfaces that are essential for PARP1 allostery. To illustrate these points, this review also includes instructive videos showing PARP1 structures and mechanisms.

Keywords:  Cancer; Chemotherapy; DNA repair; Drug resistance; PARP1; Review

DOI:  https://doi.org/10.1016/j.dnarep.2021.103125
Nucleic Acids Res. 2021 May 03. pii: gkab293. [Epub ahead of print]

The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity.

Monica Mancini, Elena Magnani, Filippo Macchi, Ian Marc Bonapace.

During S phase, the cooperation between the macromolecular complexes regulating DNA synthesis, epigenetic information maintenance and DNA repair is advantageous for cells, as they can rapidly detect DNA damage and initiate the DNA damage response (DDR). UHRF1 is a fundamental epigenetic regulator; its ability to coordinate DNA methylation and histone code is unique across proteomes of different species. Recently, UHRF1's role in DNA damage repair has been explored and recognized to be as important as its role in maintaining the epigenome. UHRF1 is a sensor for interstrand crosslinks and a determinant for the switch towards homologous recombination in the repair of double-strand breaks; its loss results in enhanced sensitivity to DNA damage. These functions are finely regulated by specific post-translational modifications and are mediated by the SRA domain, which binds to damaged DNA, and the RING domain. Here, we review recent studies on the role of UHRF1 in DDR focusing on how it recognizes DNA damage and cooperates with other proteins in its repair. We then discuss how UHRF1's epigenetic abilities in reading and writing histone modifications, or its interactions with ncRNAs, could interlace with its role in DDR.

DOI: https://doi.org/10.1093/nar/gkab293
Mol Biol. 2021 ;55(2): 211-224

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

A A Kuznetsova, A A Gavrilova, D S Novopashina, O S Fedorova, N A Kuznetsov.

  Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

Keywords:  APE1; RNA substrates; active site; endoribonuclease activity; human apurine/apyrimidine endonuclease; site-directed mutagenesis

DOI:  https://doi.org/10.1134/S0026893321020102
Front Mol Biosci. 2021 ;8 655786

Sweet Melody or Jazz? Transcription Around DNA Double-Strand Breaks.

Qilin Long, Zhichao Liu, Monika Gullerova.

  Genomic integrity is continuously threatened by thousands of endogenous and exogenous damaging factors. To preserve genome stability, cells developed comprehensive DNA damage response (DDR) pathways that mediate the recognition of damaged DNA lesions, the activation of signaling cascades, and the execution of DNA repair. Transcription has been understood to pose a threat to genome stability in the presence of DNA breaks. Interestingly, accumulating evidence in recent years shows that the transient transcriptional activation at DNA double-strand break (DSB) sites is required for efficient repair, while the rest of the genome exhibits temporary transcription silencing. This genomic shut down is a result of multiple signaling cascades involved in the maintenance of DNA/RNA homeostasis, chromatin stability, and genome fidelity. The regulation of transcription of protein-coding genes and non-coding RNAs has been extensively studied; however, the exact regulatory mechanisms of transcription at DSBs remain enigmatic. These complex processes involve many players such as transcription-associated protein complexes, including kinases, transcription factors, chromatin remodeling complexes, and helicases. The damage-derived transcripts themselves also play an essential role in DDR regulation. In this review, we summarize the current findings on the regulation of transcription at DSBs and discussed the roles of various accessory proteins in these processes and consequently in DDR.

Keywords:  cohesin; double strand break; helicase; transcription; transcription factors

DOI:  https://doi.org/10.3389/fmolb.2021.655786
Front Cell Dev Biol. 2021 ;9 663411

USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors.

Ying Zhang, Imke K Mandemaker, Syota Matsumoto, Oded Foreman, Christopher P Holland, Whitney R Lloyd, Kaoru Sugasawa, Wim Vermeulen, Jurgen A Marteijn, Paul J Galardy.

  Nucleotide excision repair (NER) is a pathway involved in the repair of a variety of potentially mutagenic lesions that distort the DNA double helix. The ubiquitin E3-ligase complex UV-DDB is required for the recognition and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) lesions through NER. DDB2 directly binds CPDs and subsequently undergoes ubiquitination and proteasomal degradation. DDB2 must remain on damaged chromatin, however, for sufficient time to recruit and hand-off lesions to XPC, a factor essential in the assembly of downstream repair components. Here we show that the tumor suppressor USP44 directly deubiquitinates DDB2 to prevent its premature degradation and is selectively required for CPD repair. Cells lacking USP44 have impaired DDB2 accumulation on DNA lesions with subsequent defects in XPC retention. The physiological importance of this mechanism is evident in that mice lacking Usp44 are prone to tumors induced by NER lesions introduced by DMBA or UV light. These data reveal the requirement for highly regulated ubiquitin addition and removal in the recognition and repair of helix-distorting DNA damage and identify another mechanism by which USP44 protects genomic integrity and prevents tumors.

Keywords:  DNA damage; animal model; deubiquitinating enzyme; tumor supperssor gene; ubiquitin

DOI:  https://doi.org/10.3389/fcell.2021.663411
J Vis Exp. 2021 Apr 16.

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1.

Miriam Yagüe-Capilla, Sean G Rudd.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

DOI: https://doi.org/10.3791/62503
Nucleic Acids Res. 2021 May 08. pii: gkab299. [Epub ahead of print]

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Wanjuan Feng, Dennis A Simpson, Jang-Eun Cho, Juan Carvajal-Garcia, Chelsea M Smith, Kathryn M Headley, Nate Hathaway, Dale A Ramsden, Gaorav P Gupta.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

DOI: https://doi.org/10.1093/nar/gkab299
Biochem J. 2021 May 07. pii: BCJ20210017. [Epub ahead of print]

Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4.

Hala Ouzon-Shubeita, Lillian Feigang Schmaltz, Seongmin Lee.

  G:T mismatches, the major mispairs generated during DNA metabolism, are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). Mismatch-specific DNA glycosylases must discriminate the mismatches against million-fold excess correct base pairs. MBD4 efficiently removes thymine opposite guanine but not opposite adenine. Previous studies have revealed that the substrate thymine is flipped out and enters the catalytic site of the enzyme, while the estranged guanine is stabilized by Arg468 of MBD4. To gain further insights into mismatch discrimination mechanism of MBD4, we assessed the glycosylase activity of MBD4 toward various base pairs. In addition, we determined a crystal structure of MBD4 bound to T:O6-methylguanine-containing DNA, which suggests the O6 and N2 of purine and the O4 of pyrimidine are required to be a substrate for MBD4. To understand the role of the Arg468 finger in catalysis, we evaluated the glycosylase activity of MBD4 mutants, which revealed the guanidinium moiety of Arg468 may play an important role in catalysis. D560N/R468K MBD4 bound to T:G mismatched DNA shows that the side chain amine moiety of the Lys stabilizes the flipped-out thymine by a water-mediated phosphate pinching, while the backbone carbonyl oxygen of the Lys engages in hydrogen bonds with N2 of the estranged guanine. Comparison of various DNA glycosylase structures implies the guanidinium and amine moieties of Arg and Lys, respectively, may involve in discriminating between substrate mismatches and nonsubstrate base pairs.

Keywords:  DNA glycosylase; base excision repair; substrate recognition

DOI:  https://doi.org/10.1042/BCJ20210017
Genetics. 2020 Dec 01. 216(4): 1009-1022

Post-translational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.

Pei-Shang Wu, Elin Enervald, Angelica Joelsson, Carina Palmberg, Dorothea Rutishauser, B Martin Hällberg, Lena Ström.

  Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28. Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo. We also demonstrated that Eco1 acetylated polymerase η in vitro. Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

Keywords:  Cdc28/Cdk1; Eco1; cohesin; damage-induced cohesion; polymerase eta

DOI:  https://doi.org/10.1534/genetics.120.303494
Mol Cell. 2021 Apr 29. pii: S1097-2765(21)00315-4. [Epub ahead of print]

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

Indrajeet Ghodke, Michaela Remisova, Audrey Furst, Sinan Kilic, Bernardo Reina-San-Martin, Anna R Poetsch, Matthias Altmeyer, Evi Soutoglou.

  p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.

Keywords:  53BP1; AHNAK; DNA repair; apoptosis; cancer; cell-cycle progression; p21; p53; phase-separation; senescence

DOI:  https://doi.org/10.1016/j.molcel.2021.04.010
Cells. 2021 Apr 30. pii: 1068. [Epub ahead of print]10(5):

The Contribution of Lysosomes to DNA Replication.

Joanna Maria Merchut-Maya, Apolinar Maya-Mendoza.

  Lysosomes, acidic, membrane-bound organelles, are not only the core of the cellular recycling machinery, but they also serve as signaling hubs regulating various metabolic pathways. Lysosomes maintain energy homeostasis and provide pivotal substrates for anabolic processes, such as DNA replication. Every time the cell divides, its genome needs to be correctly duplicated; therefore, DNA replication requires rigorous regulation. Challenges that negatively affect DNA synthesis, such as nucleotide imbalance, result in replication stress with severe consequences for genome integrity. The lysosomal complex mTORC1 is directly involved in the synthesis of purines and pyrimidines to support DNA replication. Numerous drugs have been shown to target lysosomal function, opening an attractive avenue for new treatment strategies against various pathologies, including cancer. In this review, we focus on the interplay between lysosomal function and DNA replication through nucleic acid degradation and nucleotide biosynthesis and how these could be exploited for therapeutic purposes.

Keywords:  DNA synthesis; autophagy; cancer; lysosomes; mTORC1

DOI:  https://doi.org/10.3390/cells10051068
Future Oncol. 2021 May 07.

Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells.

Maryam Katoueezadeh, Niloofar Pilehvari, Ahmad Fatemi, Gholamhossein Hassanshahi, Seyedeh Atekeh Torabizadeh.

  An alarming increase in acute lymphoblastic leukemia cases among children and adults has attracted the attention of researchers to discover new therapeutic strategies with a better prognosis. In cancer cells, the DNA damage response (DDR) pathway elements have been recognized to protect tumor cells from various stresses and cause tumor progression; targeting these DDR members is an attractive strategy for treatment of cancers. The inhibition of the DDR pathway in cancer cells for the treatment of cancers has recently been introduced. Hence, effective treatment strategies are needed for this purpose. Chemotherapy in combination with radiotherapy is considered a potential therapeutic strategy for acute leukemia. This review aims to assess the synergistic effects of these inhibitors with irradiation for the treatment of leukemia.

Keywords:  DNA damage; DNA damage response; acute lymphoblastic leukemia; radiation; response pathway inhibitors

DOI:  https://doi.org/10.2217/fon-2020-1072
Mol Pharmacol. 2021 May 03. pii: MOLPHARM-AR-2021-000262. [Epub ahead of print]

A role for VCP/p97 in the processing of drug-stabilised TOP2-DNA covalent complexes.

Rebecca L Swan, Ian G Cowell, Caroline A Austin.

  DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase which utilises energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A- and TOP2B- DNA complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2AX, indicative of fewer DSBs . This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes, and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. Significance Statement TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. Better understanding how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.

Keywords:  Proteasome-mediated protein degradation; Topoisomerases

DOI:  https://doi.org/10.1124/molpharm.121.000262
Genetics. 2019 Oct 01. 213(2): 411-429

Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase.

Luca Menin, Chiara Vittoria Colombo, Giorgia Maestrini, Maria Pia Longhese, Michela Clerici.

  Mecl/ATR and Tell/ATM trigger replicative senescence when telomeres become critically short in the absence of telomerase, but how Tell/ATM promotes senescence is still unclear. Menin et al. studied the functions of Saccharomyces cerevisiae Tel1 in senescence by using... Telomeres progressively shorten at every round of DNA replication in the absence of telomerase. When they become critically short, telomeres trigger replicative senescence by activating a DNA damage response that is governed by the Mec1/ATR and Tel1/ATM protein kinases. While Mec1/ATR is known to block cell division when extended single-stranded DNA (ssDNA) accumulates at eroded telomeres, the molecular mechanism by which Tel1/ATM promotes senescence is still unclear. By characterizing a Tel1-hy184 mutant variant that compensates for the lack of Mec1 functions, we provide evidence that Tel1 promotes senescence by signaling to a Rad9-dependent checkpoint. Tel1-hy184 anticipates senescence onset in telomerase-negative cells, while the lack of Tel1 or the expression of a kinase-defective (kd) Tel1 variant delays it. Both Tel1-hy184 and Tel1-kd do not alter ssDNA generation at telomeric DNA ends. Furthermore, Rad9 and (only partially) Mec1 are responsible for the precocious senescence promoted by Tel1-hy184. This precocious senescence is mainly caused by the F1751I, D1985N, and E2133K amino acid substitutions, which are located in the FRAP-ATM-TRAPP domain of Tel1 and also increase Tel1 binding to DNA ends. Altogether, these results indicate that Tel1 induces replicative senescence by directly signaling dysfunctional telomeres to the checkpoint machinery.

Keywords:  Tel1; checkpoint; replicative senescence; telomere

DOI:  https://doi.org/10.1534/genetics.119.302391
Sci Adv. 2021 May;pii: eabf7684. [Epub ahead of print]7(19):

PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability.

Shuai Ma, Cheng Cao, Shiyou Che, Yuejiao Wang, Dongxue Su, Shuai Liu, Wenchen Gong, Ling Liu, Jixue Sun, Jiao Zhao, Qian Wang, Nan Song, Tong Ge, Qiushi Guo, Shanshan Tian, Charlie Degui Chen, Tao Zhang, Ju Wang, Xiang Ding, Fuquan Yang, Guoguang Ying, Jie Yang, Kai Zhang, Yi Zhu, Zhi Yao, Na Yang, Lei Shi.

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.

DOI: https://doi.org/10.1126/sciadv.abf7684
Genetics. 2019 Oct 01. 213(2): 449-463

Mutations in the PCNA DNA Polymerase Clamp of Saccharomyces cerevisiae Reveal Complexities of the Cell Cycle and Ploidy on Heterochromatin Assembly.

Molly Brothers, Jasper Rine.

  Proliferating Cell Nuclear Antigen (PCNA) is a DNA polymerase clamp conserved in all eukaryotes that coordinates many activities at the replication fork and at sites of DNA damage. Alleles of PCNA, encoded by POL30 in Saccharomyces cerevisiae, disrupt transcriptional... In Saccharomyces cerevisiae, transcriptional silencing at HML and HMR maintains mating-type identity. The repressive chromatin structure at these loci is replicated every cell cycle and must be re-established quickly to prevent transcription of the genes at these loci. Mutations in a component of the replisome, the proliferating cell nuclear antigen (PCNA), encoded by POL30, cause a loss of transcriptional silencing at HMR. We used an assay that captures transient losses of silencing at HML and HMR to perform extended genetic analyses of the pol30-6, pol30-8, and pol30-79 alleles. All three alleles destabilized silencing only transiently and only in cycling cells. Whereas pol30-8 caused loss of silencing by disrupting the function of Chromatin Assembly Factor 1, pol30-6 and pol30-79 acted through a separate genetic pathway, but one still dependent on histone chaperones. Surprisingly, the silencing-loss phenotypes of pol30-6 and pol30-79 depended on ploidy, but not on POL30 dosage or mating-type identity. Separately from silencing loss, the pol30-6 and pol30-79 alleles also displayed high levels of mitotic recombination in diploids. These results established that histone trafficking involving PCNA at replication forks is crucial to the maintenance of chromatin state and genome stability during DNA replication. They also raised the possibility that increased ploidy may protect chromatin states when the replisome is perturbed.

Keywords:   POL30 ; intragenic complementation; nucleosome assembly; recombination; transcriptional silencing

DOI:  https://doi.org/10.1534/genetics.119.302452
Front Pharmacol. 2021 ;12 653549

Unfolding New Roles for Guanine-Based Purines and Their Metabolizing Enzymes in Cancer and Aging Disorders.

P Di Iorio, S Beggiato, M Ronci, C B Nedel, C I Tasca, M Zuccarini.



Keywords:  aging disease; cancer; guanine deaminase; guanine-based purines; purine nucleoside phosphorylase; reactive oxygen species

DOI:  https://doi.org/10.3389/fphar.2021.653549
Mol Cell. 2021 Apr 29. pii: S1097-2765(21)00234-3. [Epub ahead of print]

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

Nishita Parnandi, Veronica Rendo, Gaofeng Cui, Maria Victoria Botuyan, Michaela Remisova, Huy Nguyen, Pascal Drané, Rameen Beroukhim, Matthias Altmeyer, Georges Mer, Dipanjan Chowdhury.

  53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

Keywords:  53BP1; NMR; TIRR; Tudor; cancer; cell fate; p53; senescence; survival; transcription factor

DOI:  https://doi.org/10.1016/j.molcel.2021.03.039
Genetics. 2019 Nov 01. 213(3): 805-818

Unresolved Recombination Intermediates Cause a RAD9-Dependent Cell Cycle Arrest in Saccharomyces cerevisiae.

Hardeep Kaur, Krishnaprasad Gn, Michael Lichten.

  It has been suggested that the conserved Sgs1-Top3-Rmi1 (STR) helicasedecatenase complex resolves double Holliday junction recombination intermediates (dHJs) as noncrossovers by a process called dissolution. Lichten, Kaur, and GN tested this by accumulating dHJs during meiosis... In Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are crossover precursors. In vitro studies have suggested that this may be due to dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation. To ask whether dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth (RTG), a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during RTG delayed joint molecule resolution, but, ultimately, most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9∆ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in Rmi1-depleted rad9∆ cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

Keywords:  DNA damage checkpoint; Sgs1-Top3-Rmi1 helicase-decatenase; double Holliday junction dissolution; homologous recombination; mitotic cell cycle

DOI:  https://doi.org/10.1534/genetics.119.302632
Genetics. 2019 Jul 01. 212(3): 631-654

Tolerance of DNA Replication Stress Is Promoted by Fumarate Through Modulation of Histone Demethylation and Enhancement of Replicative Intermediate Processing in Saccharomyces cerevisiae.

Faeze Saatchi, Ann L Kirchmaier.

  Genome integrity is fundamental to viability and health and can be impacted by metabolic alterations that affect chromatin composition. Saatchi and Kirchmaier present evidence that loss of fumarase, an ortholog of the tumor suppressor... Fumarase is a well-characterized TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate. In mammals, fumarase acts as a tumor suppressor, and loss-of-function mutations in the FH gene in hereditary leiomyomatosis and renal cell cancer result in the accumulation of intracellular fumarate-an inhibitor of α-ketoglutarate-dependent dioxygenases. Fumarase promotes DNA repair by nonhomologous end joining in mammalian cells through interaction with the histone variant H2A.Z, and inhibition of KDM2B, a H3 K36-specific histone demethylase. Here, we report that Saccharomyces cerevisiae fumarase, Fum1p, acts as a response factor during DNA replication stress, and fumarate enhances survival of yeast lacking Htz1p (H2A.Z in mammals). We observed that exposure to DNA replication stress led to upregulation as well as nuclear enrichment of Fum1p, and raising levels of fumarate in cells via deletion of FUM1 or addition of exogenous fumarate suppressed the sensitivity to DNA replication stress of htz1Δ mutants. This suppression was independent of modulating nucleotide pool levels. Rather, our results are consistent with fumarate conferring resistance to DNA replication stress in htz1Δ mutants by inhibiting the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation. Although the timing of checkpoint activation and deactivation remained largely unaffected by fumarate, sensors and mediators of the DNA replication checkpoint were required for fumarate-dependent resistance to replication stress in the htz1Δ mutants. Together, our findings imply metabolic enzymes and metabolites aid in processing replicative intermediates by affecting chromatin modification states, thereby promoting genome integrity.

Keywords:   HTZ1 ; JHD2 ; DNA replication stress; fumarate; histone methylation

DOI:  https://doi.org/10.1534/genetics.119.302238
Nucleic Acids Res. 2021 May 06. pii: gkab282. [Epub ahead of print]

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion.

Pedro Silva-Pinheiro, Carlos Pardo-Hernández, Aurelio Reyes, Lisa Tilokani, Anup Mishra, Raffaele Cerutti, Shuaifeng Li, Dieu-Hien Rozsivalova, Sebastian Valenzuela, Sukru A Dogan, Bradley Peter, Patricio Fernández-Silva, Aleksandra Trifunovic, Julien Prudent, Michal Minczuk, Laurence Bindoff, Bertil Macao, Massimo Zeviani, Maria Falkenberg, Carlo Viscomi.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

DOI: https://doi.org/10.1093/nar/gkab282
Nucleic Acids Res. 2021 May 08. pii: gkab239. [Epub ahead of print]

Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches.

Dian Spakman, Julia A M Bakx, Andreas S Biebricher, Erwin J G Peterman, Gijs J L Wuite, Graeme A King.

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes.

DOI: https://doi.org/10.1093/nar/gkab239
J Vis Exp. 2021 Apr 16.

Laser Micro-Irradiation to Study DNA Recruitment During S Phase.

Bearach Miwatani-Minter, Gergely Rona.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

DOI: https://doi.org/10.3791/62466
Elife. 2021 May 04. pii: e58741. [Epub ahead of print]10

Reduced purine biosynthesis in humans after their divergence from Neandertals.

Vita Stepanova, Kaja Ewa Moczulska, Guido Vacano, Ilia Kurochkin, Xiangchun Ju, Stephan Riesenberg, Dominik Macak, Tomislav Maricic, Linda Dombrowski, Maria Schörnig, Konstantinos Anastassiadis, Oliver Baker, Ronald Naumann, Ekaterina Khrameeva, Anna Vanushkina, Elena Stekolshchikova, Alina Egorova, Anna Tkachev, Randall Mazzarino, Nathan Duval, Dmitri Zubkov, Patrick Giavalisco, Terry G Wilkinson Ii, David Patterson, Philipp Khaitovich, Svante Pääbo.

  We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Keywords:  evolutionary biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.58741
Trends Cell Biol. 2021 May 04. pii: S0962-8924(21)00072-6. [Epub ahead of print]

REV7: Jack of many trades.

Inge de Krijger, Vera Boersma, Jacqueline J L Jacobs.

  The HORMA domain protein REV7, also known as MAD2L2, interacts with a variety of proteins and thereby contributes to the establishment of different complexes. With doing so, REV7 impacts a diverse range of cellular processes and gained increasing interest as more of its activities became uncovered. REV7 has important roles in translesion synthesis and mitotic progression, and acts as a central component in the recently discovered shieldin complex that operates in DNA double-strand break repair. Here we discuss the roles of REV7 in its various complexes, focusing on its activity in genome integrity maintenance. Moreover, we will describe current insights on REV7 structural features that allow it to be such a versatile protein.

Keywords:  HORMA; REV7; genome maintenance; shieldin; translesion synthesis

DOI:  https://doi.org/10.1016/j.tcb.2021.04.002
Mol Cancer Ther. 2021 May 04. pii: molcanther.MCT-20-0252-E.2020. [Epub ahead of print]

Combined inhibition of Rad51 and Wee1 enhances cell killing in HNSCC through induction of apoptosis associated with excessive DNA damage and replication stress.

Jeffrey N Myers, Antje Lindemann, Ameeta A Patel, Lin Tang, Noriaki Tanaka, Frederico O Gleber-Netto, Mason D Bartels, Li Wang, Daniel J McGrail, Shiaw-Yih Lin, Steven J Frank, Mitchell J Frederick, Abdullah A Osman.

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo. In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0252
Cancer Res. 2021 May 03. pii: canres.3309.2020. [Epub ahead of print]

Disabling the Fanconi Anemia Pathway in Stem Cells Leads to Radioresistance and Genomic Instability.

Xinzhu Deng, Jason Tchieu, Daniel S Higginson, Kuo-Shun Hsu, Regina Feldman, Lorenz Studer, Shai Shaham, Simon N Powell, Zvi Fuks, Richard Kolesnick.

Fanconi Anemia (FA) is an inherited genome instability syndrome characterized by interstrand crosslink hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by FA genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with FA is not well understood. Here we report that FA stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)-dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. By contrast, DNA crosslinking does not induce generational genomic instability in FA stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that FA is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3309
FEBS J. 2021 May 06.

Loss of BubR1 acetylation provokes replication stress and leads to complex chromosomal rearrangements.

Jiho Park, Song-Yion Yeu, Sangjin Paik, Hyungmin Kim, Si-Young Choi, Junyeob Lee, Jinho Jang, Semin Lee, Young-Il Koh, Hyunsook Lee.

  Accurate chromosome segregation during mitosis is regulated by the spindle assembly checkpoint (SAC). SAC failure results in aneuploidy, a hallmark of cancer. However, many studies have suggested that aneuploidy alone is not oncogenic. We have reported that BubR1 acetylation deficiency in mice (K243R/+) caused spontaneous tumorigenesis via weakened SAC signaling and unstable chromosome-spindle attachment, resulting in massive chromosome mis-segregation. In addition to aneuploidy, cells derived from K243R/+ mice exhibited moderate genetic instability and chromosomal translocation. Here, we investigated how the loss of BubR1 acetylation led to genetic instability and chromosome rearrangement. To rescue all chromosomal abnormalities generated by the loss of BubR1 acetylation during development, K243R/+ mice were crossed with p53-deficient mice. Genome-wide sequencing and spectral karyotyping of tumors derived from these double-mutant mice revealed that BubR1 acetylation deficiency was associated with complex chromosomal rearrangements, including Robertsonian-like whole-arm translocations. By analyzing the telomeres and centromeres in metaphase chromosome spreads, we found that BubR1 acetylation deficiency increased the collapse of stalled replication forks, commonly referred to as replication stress, and led to DNA damage and chromosomal rearrangements. BubR1 mutations L249F and A251P, which are critical for interaction with PCAF acetyltransferase and for acetylation of K250, were found in human cancer and a subset of human cancer cell lines. Notably, these samples exhibited whole-arm translocation and displayed defects in BubR1 acetylation as well, supporting that defects in BubR1 acetylation in mitosis contributes to tumorigenesis. Collectively, loss of BubR1 acetylation provokes replication stress, particularly at the telomeres, leading to genetic instability and chromosomal rearrangement.

Keywords:  BubR1 acetylation; cancer; chromosome instability; p53; replication stress

DOI:  https://doi.org/10.1111/febs.15912