bims-numges 2021-02-07 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒02‒07
38 papers selected by
Sean Rudd
Karolinska Institutet

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.
The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure.
To skip or not to skip: choosing repriming to tolerate DNA damage.
The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.
A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol.
DNA replication stress and emerging prospects for PARG inhibitors in ovarian cancer therapy.
SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors.
PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.
DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities.
Cellular response to endogenous DNA damage: DNA base modifications in gene expression regulation.
Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair.
Exogenous and endogenous sources of serine contribute to colon cancer metabolism, growth, and resistance to 5-fluorouracil.
Nuclear translocation of the receptor tyrosine kinase c-MET reduces the treatment efficacies of olaparib and gemcitabine in pancreatic ductal adenocarcinoma cells.
Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment.
RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.
Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis.
DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment.
Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes.
Targeting DNA damage repair functions of two histone deacetylases, HDAC8 and SIRT6, sensitizes acute myeloid leukemia to NAMPT inhibition.
Targeting DNA Damage Response Pathways to Activate the STING Innate Immune Signaling Pathway in Human Cancer Cells.
Cell cycle inertia underlies a bifurcation in cell fates after DNA damage.
The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers.
Identification of Key Genes in Purine Metabolism as Prognostic Biomarker for Hepatocellular Carcinoma.
Deeper Insight in Metastatic Cancer progression;Epithelial-to-Mesenchymal Transition and Genomic Instability: implications on treatment resistance.
Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.
The multifaceted roles of DNA repair and replication proteins in aging and obesity.
Co-therapy with S-adenosylmethionine and nicotinamide riboside improves t-cell survival and function in Arts Syndrome (PRPS1 deficiency).
Development of MTH1-Binding Nucleotide Analogs Based on 7,8-Dihalogenated 7-Deaza-dG Derivatives.
A conserved histone H3-H4 interface regulates DNA damage tolerance and homologous recombination during the recovery from replication stress.
The Role of DNA Polymerase ζ in Benzo[a]pyrene-induced Mutagenesis in the Mouse Lung.
APE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling.
Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.
Damage-induced chromatome dynamics link Ubiquitin ligase and proteasome recruitment to histone loss and efficient DNA repair.
Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase.
Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.
Design, Synthesis and Biological Evaluation of New Pyrimidine Derivatives as Anticancer Agents.
Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.
Expression of the novel tumour suppressor sterile alpha motif and HD domain-containing protein 1 is an independent adverse prognostic factor in classical Hodgkin lymphoma.

Mol Cell. 2021 Feb 04. pii: S1097-2765(21)00004-6. [Epub ahead of print]81(3): 426-441.e8

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Mathew J K Jones, Camille Gelot, Stephanie Munk, Amnon Koren, Yoshitaka Kawasoe, Kelly A George, Ruth E Santos, Jesper V Olsen, Steven A McCarroll, Mark G Frattini, Tatsuro S Takahashi, Prasad V Jallepalli.

  Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

Keywords:  ATR; Cdc7; DDK; DNA replication; MERIT40; PDS5B; chemical genetics; phosphoproteomics

DOI:  https://doi.org/10.1016/j.molcel.2021.01.004
Elife. 2021 Feb 01. pii: e61797. [Epub ahead of print]10

The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure.

Hsiang-Chen Chou, Kuhulika Bhalla, Osama El Demerdesh, Olaf Klingbeil, Kaarina Hanington, Sergey Aganezov, Peter Andrews, Habeeb Alsudani, Kenneth Chang, Christopher R Vakoc, Michael C Schatz, W Richard McCombie, Bruce Stillman.

  The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

Keywords:  chromosomes; gene expression; human

DOI:  https://doi.org/10.7554/eLife.61797
Mol Cell. 2021 Jan 21. pii: S1097-2765(21)00012-5. [Epub ahead of print]

To skip or not to skip: choosing repriming to tolerate DNA damage.

Annabel Quinet, Stephanie Tirman, Emily Cybulla, Alice Meroni, Alessandro Vindigni.

Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.

DOI: https://doi.org/10.1016/j.molcel.2021.01.012
Mol Cell. 2021 Feb 04. pii: S1097-2765(20)30829-7. [Epub ahead of print]81(3): 442-458.e9

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Irene Gallina, Ivo A Hendriks, Saskia Hoffmann, Nicolai B Larsen, Joachim Johansen, Camilla S Colding-Christensen, Lisa Schubert, Selene Sellés-Baiget, Zita Fábián, Ulrike Kühbacher, Alan O Gao, Markus Räschle, Simon Rasmussen, Michael L Nielsen, Niels Mailand, Julien P Duxin.

  Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

Keywords:  CHROMASS; DPC repair; HMCES; ICL repair; PCNA; RFWD3; TLS; UV lesions; anemia; ubiquitylation

DOI:  https://doi.org/10.1016/j.molcel.2020.11.029
Nucleic Acids Res. 2021 Feb 03. pii: gkab049. [Epub ahead of print]

A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol.

Kazutoshi Kasho, Gorazd Stojkovič, Cristina Velázquez-Ruiz, Maria Isabel Martínez-Jiménez, Mara Doimo, Timothée Laurent, Andreas Berner, Aldo E Pérez-Rivera, Louise Jenninger, Luis Blanco, Sjoerd Wanrooij.

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.

DOI: https://doi.org/10.1093/nar/gkab049
Prog Biophys Mol Biol. 2021 Jan 29. pii: S0079-6107(21)00005-5. [Epub ahead of print]

DNA replication stress and emerging prospects for PARG inhibitors in ovarian cancer therapy.

Nisha Pillay, Rosie Brady, Malini Dey, Robert D Morgan, Stephen S Taylor.

  Poly (ADP-ribosyl)ation has central functions in maintaining genome stability, including facilitating DNA replication and repair. In cancer cells these processes are frequently disrupted, and thus interfering with poly (ADP-ribosyl)ation can exacerbate inherent genome instability and induce selective cytotoxicity. Indeed, inhibitors of poly (ADP-ribose) polymerase (PARP) are having a major clinical impact in treating women with BRCA-mutant ovarian cancer, based on a defect in homologous recombination. However, only around half of ovarian cancers harbour defects in homologous recombination, and most sensitive tumours eventually acquire PARP inhibitor resistance with treatment. Thus, there is a pressing need to develop alternative treatment strategies to target tumours with both inherent and acquired resistance to PARP inhibition. Several novel inhibitors of poly (ADP-ribose)glycohydrolase (PARG) have been described, with promising anti-cancer activity in vitro that is distinct from PARP inhibitors. Here we discuss, the role of poly (ADP-ribosyl)ation in genome stability, and the potential for PARG inhibitors as a complementary strategy to PARP inhibitors in the treatment of ovarian cancer.

Keywords:  Ovarian cancer; Poly(ADP-ribos)ylation; Poly(ADP-ribose)glycohydolase; Replication stress

DOI:  https://doi.org/10.1016/j.pbiomolbio.2021.01.004
Proc Natl Acad Sci U S A. 2021 Feb 09. pii: e2015654118. [Epub ahead of print]118(6):

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors.

Ukhyun Jo, Yasuhisa Murai, Sirisha Chakka, Lu Chen, Ken Cheng, Junko Murai, Liton Kumar Saha, Lisa M Miller Jenkins, Yves Pommier.

  Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.

Keywords:  ATR/CHK1 inhibitor; CDT1; CUL4; SLFN11

DOI:  https://doi.org/10.1073/pnas.2015654118
J Biol Chem. 2020 Jan 03. pii: S0021-9258(17)49556-1. [Epub ahead of print]295(1): 146-157

PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.

Carmen Morales, Miguel Ruiz-Torres, Sara Rodríguez-Acebes, Vanesa Lafarga, Miriam Rodríguez-Corsino, Diego Megías, David A Cisneros, Jan-Michael Peters, Juan Méndez, Ana Losada.

  Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.

Keywords:  BRCA2; DNA replication; cell cycle; cohesin; fork protection; fork reversal; fork stalling; genomic instability; microscopy; replication stress; replisome

DOI:  https://doi.org/10.1074/jbc.RA119.011099
DNA Repair (Amst). 2021 Jan 21. pii: S1568-7864(21)00004-5. [Epub ahead of print]99 103050

DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities.

Beverly A Baptiste, Stephanie L Baringer, Tomasz Kulikowicz, Joshua A Sommers, Deborah L Croteau, Robert M Brosh, Vilhelm A Bohr.

  DNA polymerase beta (POLβ), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLβ and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLβ is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLβ has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLβ are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLβ may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

Keywords:  Ber; Mitochondria; Polb; Polg

DOI:  https://doi.org/10.1016/j.dnarep.2021.103050
DNA Repair (Amst). 2021 Jan 26. pii: S1568-7864(21)00005-7. [Epub ahead of print]99 103051

Cellular response to endogenous DNA damage: DNA base modifications in gene expression regulation.

Diana L Bordin, Lisa Lirussi, Hilde Nilsen.

  The integrity of the genetic information is continuously challenged by numerous genotoxic insults, most frequently in the form of oxidation, alkylation or deamination of the bases that result in DNA damage. These damages compromise the fidelity of the replication, and interfere with the progression and function of the transcription machineries. The DNA damage response (DDR) comprises a series of strategies to deal with DNA damage, including transient transcriptional inhibition, activation of DNA repair pathways and chromatin remodeling. Coordinated control of transcription and DNA repair is required to safeguardi cellular functions and identities. Here, we address the cellular responses to endogenous DNA damage, with a particular focus on the role of DNA glycosylases and the Base Excision Repair (BER) pathway, in conjunction with the DDR factors, in responding to DNA damage during the transcription process. We will also discuss functions of newly identified epigenetic and regulatory marks, such as 5-hydroxymethylcytosine and its oxidative products and 8-oxoguanine, that were previously considered only as DNA damages. In light of these resultsthe classical perception of DNA damage as detrimental for cellular processes are changing. and a picture emerges whereDNA glycosylases act as dynamic regulators of transcription, placing them at the intersection of DNA repair and gene expression modulation.

Keywords:  Base excision repair; DNA damage response; DNA glycosylases; Endogenous DNA damage; Epigenetic marks; Transcription regulation

DOI:  https://doi.org/10.1016/j.dnarep.2021.103051
Nucleic Acids Res. 2021 Feb 03. pii: gkab019. [Epub ahead of print]

Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair.

Laura A Murray-Nerger, Joshua L Justice, Pranav Rekapalli, Josiah E Hutton, Ileana M Cristea.

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

DOI: https://doi.org/10.1093/nar/gkab019
Cancer Res. 2021 Feb 01. pii: canres.1541.2020. [Epub ahead of print]

Exogenous and endogenous sources of serine contribute to colon cancer metabolism, growth, and resistance to 5-fluorouracil.

David C Montrose, Suchandrima Saha, Miguel Foronda, Erin M McNally, Justin Chen, Xi Kathy Zhou, Taehoon Ha, Jan Krumsiek, Mustafa Buyukozkan, Akanksha Verma, Olivier Elemento, Rhonda K Yantiss, Qiuying Chen, Steven S Gross, Lorenzo Galluzzi, Lukas E Dow, Andrew J Dannenberg.

Serine is a non-essential amino acid generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. Additionally, cancer cells can harness exogenous serine to enhance their metabolism and proliferation. Here we tested the relative contributions of exogenous and endogenous sources of serine on the biology of colorectal cancer (CRC). In murine tumors, Apc status was identified as a determinant of the expression of genes controlling serine synthesis. In patient samples, PSAT1 was overexpressed in both colorectal adenomas and adenocarcinomas. Combining genetic deletion of PSAT1 with exogenous serine deprivation maximally suppressed the proliferation of CRC cells and induced profound metabolic defects including diminished nucleotide production. Inhibition of serine synthesis enhanced the transcriptional changes following exogenous serine removal as well as alterations associated with DNA damage. Both loss of PSAT1 and removal of serine from the diet were necessary to suppress CRC xenograft growth and enhance the anti-tumor activity of 5-fluorouracil (5-FU). Restricting endogenous and exogenous serine in vitro augmented 5-FU induced cell death, DNA damage, and metabolic perturbations, likely accounting for the observed anti-tumor effect. Collectively, our results suggest that both endogenous and exogenous sources of serine contribute to CRC growth and resistance to 5-FU.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-1541
Am J Cancer Res. 2021 ;11(1): 236-250

Nuclear translocation of the receptor tyrosine kinase c-MET reduces the treatment efficacies of olaparib and gemcitabine in pancreatic ductal adenocarcinoma cells.

Yuan Gao, Mei-Kuang Chen, Yu-Yi Chu, Liuqing Yang, Dihua Yu, Yingbin Liu, Mien-Chie Hung.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that lack effective therapeutic strategies. The response rate of PDAC for treatment with gemcitabine, a current first-line chemotherapeutic for this tumor, is lower than 20%. Identifying key targetable molecules that mediate gemcitabine resistance and developing novel strategies for precision PDAC medicine are urgently needed. Most PDACs have either intratumoral hypoxia or high reactive oxygen species (ROS) production; cytotoxic chemotherapy can elevate ROS production in PDACs. Although excessive ROS production leads to oxidative damage of macromolecules such as DNA, pancreatic cancer cells can survive high DNA damage stress levels. Therefore, identifying molecular mechanisms of overcoming ROS-induced stress in pancreatic cancer cells is important for developing novel therapeutic strategies. ROS-induced DNA damage is predominantly repaired via poly (ADP-ribose) polymerase 1 (PARP1)-mediated DNA repair mechanisms. A recent clinical trial reported that PARP inhibitors are effective in treating pancreatic patients carrying BRCA mutations. However, only less than 10% of pancreatic cancer patients bearing BRCA mutated tumors. Activation of the receptor tyrosine kinase c-MET positively correlates with poor prognosis for PDAC, and our previous study showed that nuclear c-MET can phosphorylate PARP1 at tyrosine 907 under ROS stimulation to promote DNA repair. As described herein, we proposed to expand PARP inhibitor-targeted therapy to more pancreatic cancer patients regardless of BRCA mutation status by combining olaparib, a PARP inhibitor, with c-MET inhibitors as we demonstrated in our previous studies in breast cancer. In this prospective study, we found that ROS-inducing chemotherapeutic drugs such as gemcitabine and doxorubicin stimulated nuclear accumulation of c-MET in BxPC-3 and L3.6pl pancreatic cancer cells. We further showed that combining a c-MET inhibitor with gemcitabine or a PARP inhibitor induced more DNA damage than monotherapy did. Moreover, we demonstrated the synergistic antitumor effects of c-MET inhibitors combined with a PARP inhibitor or gemcitabine in eliminating pancreatic cancer cells. These data suggested that accumulation of ROS in pancreatic cancer cells promotes nuclear localization of c-MET, resulting in resistance to both chemotherapy and PARP inhibitors. Our findings suggest that combining c-MET inhibitors with PARP inhibitors or gemcitabine is a novel, rational therapeutic strategy for advanced pancreatic cancer.

Keywords: DNA damage; PARP inhibitor; Pancreatic ductal adenocarcinoma; chemotherapy; precision medicine; reactive oxygen species; resistance; targeted therapy; tivantinib
J Biol Chem. 2021 Feb 01. pii: S0021-9258(21)00127-7. [Epub ahead of print] 100355

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment.

David R McKinzey, Shivasankari Gomathinayagam, Wezley C Griffin, Kathleen N Klinzing, Elizabeth P Jeffries, Aleksandar Rajkovic, Michael A Trakselis.

  The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC) induced DNA damage. First, an unconventional 'bipartite-like' nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full length MCM9 for proper DNA repair.

Keywords:  BRC variant motif; DNA repair; MCM9; NLS; Rad51; homologous recombination; mitomycin C

DOI:  https://doi.org/10.1016/j.jbc.2021.100355
J Clin Invest. 2021 Feb 01. pii: 140105. [Epub ahead of print]131(3):

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.

Parasvi S Patel, Karan Joshua Abraham, Kiran Kumar Naidu Guturi, Marie-Jo Halaby, Zahra Khan, Luis Palomero, Brandon Ho, Shili Duan, Jonathan St-Germain, Arash Algouneh, Francesca Mateo, Samah El Ghamrasni, Haithem Barbour, Daniel R Barnes, Jonathan Beesley, Otto Sanchez, Hal K Berman, Grant W Brown, El Bachir Affar, Georgia Chenevix-Trench, Antonis C Antoniou, Cheryl H Arrowsmith, Brian Raught, Miquel Angel Pujana, Karim Mekhail, Anne Hakem, Razqallah Hakem.

  Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Keywords:  Breast cancer; Cell Biology; Molecular genetics; Mouse models; Oncology

DOI:  https://doi.org/10.1172/JCI140105
Sci Adv. 2021 Jan;pii: eabb5414. [Epub ahead of print]7(1):

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis.

Anna Gueiderikh, Frédérique Maczkowiak-Chartois, Guillaume Rouvet, Sylvie Souquère-Besse, Sébastien Apcher, Jean-Jacques Diaz, Filippo Rosselli.

Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex-independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

DOI: https://doi.org/10.1126/sciadv.abb5414
Cancers (Basel). 2021 Jan 28. pii: 504. [Epub ahead of print]13(3):

DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment.

Takayuki Saitoh, Tsukasa Oda.

  Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

Keywords:  DNA damage response; DNA repair; base excision repair; genomic instability; homologous recombination; multiple myeloma

DOI:  https://doi.org/10.3390/cancers13030504
J Biol Chem. 2020 Jul 03. pii: S0021-9258(17)50324-5. [Epub ahead of print]295(27): 9012-9020

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes.

Carel Fijen, Mariam M Mahmoud, Meike Kronenberg, Rebecca Kaup, Mattia Fontana, Jamie B Towle-Weicksel, Joann B Sweasy, Johannes Hohlbein.

  Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Keywords:  DNA binding; DNA polymerase; base excision repair; conformational change; fluorescence resonance energy transfer (FRET); genome integrity; single-molecule analysis; substrate specificity

DOI:  https://doi.org/10.1074/jbc.RA120.013049
Clin Cancer Res. 2021 Feb 04. pii: clincanres.3724.2020. [Epub ahead of print]

Targeting DNA damage repair functions of two histone deacetylases, HDAC8 and SIRT6, sensitizes acute myeloid leukemia to NAMPT inhibition.

Pu Zhang, Lindsey T Brinton, Katie Williams, Steven Sher, Shelley Orwick, Tzung-Huei Lai, Alice S Mims, Christopher C Coss, Samuel K Kulp, Youssef Youssef, Wing Keung Chan, Shaneice Mitchell, Allison Mustonen, Matthew Cannon, Hannah Phillips, Amy M Lehman, Tierney Kauffman, Larry Beaver, Daniel Canfield, Nicole R Grieselhuber, Lapo Alinari, Deepa Sampath, Pearlly Yan, John C Byrd, James S Blachly, Rosa Lapalombella.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors are currently in development, but may be limited as a single agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPT inhibitors required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPT inhibitor, KPT-2974.EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model.
RESULTS: We identified two histone deacetylases, HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical Class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 co-treatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 co-treatment resulted in synergistic attenuation of homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways in cell lines and leukemia initiating cells (LICs).
CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity towards normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

DOI: https://doi.org/10.1158/1078-0432.CCR-20-3724
FEBS J. 2021 Feb 02.

Targeting DNA Damage Response Pathways to Activate the STING Innate Immune Signaling Pathway in Human Cancer Cells.

Joanne Wayne, Teresa Brooks, Alexandra Landras, Andrew J Massey.

  Activating STING to turn immunologically refractive cold tumors hot is an exciting therapeutic approach to increase the clinical responsiveness of some human cancers to immune checkpoint inhibitors. DNA damaging drugs and PARP inhibitors are two types of agents that have demonstrated this potential. Inhibitors of Chk1 or Wee1 induce DNA damage in cancer cells in predominantly the S-phase population. Increased cytoplasmic ss and dsDNA from this DNA damage resulted in increased TBK1 phosphorylation in a range of cancer cell lines. However, despite robust increases in pTBK1, no downstream consequences of TBK1 phosphorylation were observed (namely no increase in pIRF3/7, IRF-dependent gene expression or a Type I IFN response). In combination with cytotoxic chemotherapy such as gemcitabine or camptothecin, Chk1 inhibition increased cytoplasmic dsDNA compared to the cytotoxic alone but attenuated the cytotoxic chemotherapy-induced increase in IRF1 protein and STAT1 phosphorylation through inhibition of nuclear RelB translocation. Despite increased cytoplasmic DNA and TBK1 activation, inhibition of Chk1, ATR or Wee1 failed to activate a type I IFN response. We discuss the potential underlying mechanisms for this lack of IRF-dependent gene response and how this might influence the clinical strategies of combining Chk1 or Wee1 inhibitors with immune checkpoint inhibitors.

Keywords:  ATR; Chk1; DNA damage response; STING; Wee1; cytoplasmic DNA; innate immune response

DOI:  https://doi.org/10.1111/febs.15747
Sci Adv. 2021 Jan;pii: eabe3882. [Epub ahead of print]7(3):

Cell cycle inertia underlies a bifurcation in cell fates after DNA damage.

Jenny F Nathans, James A Cornwell, Marwa M Afifi, Debasish Paul, Steven D Cappell.

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

DOI: https://doi.org/10.1126/sciadv.abe3882
Sci Rep. 2021 Feb 04. 11(1): 3176

The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers.

Stijn Moens, Peihua Zhao, Maria Francesca Baietti, Oliviero Marinelli, Delphi Van Haver, Francis Impens, Giuseppe Floris, Elisabetta Marangoni, Patrick Neven, Daniela Annibali, Anna A Sablina, Frédéric Amant.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs.

DOI: https://doi.org/10.1038/s41598-021-82780-6
Front Oncol. 2020 ;10 583053

Identification of Key Genes in Purine Metabolism as Prognostic Biomarker for Hepatocellular Carcinoma.

Wen-Jing Su, Pei-Zhi Lu, Yong Wu, Kumari Kalpana, Cheng-Kun Yang, Guo-Dong Lu.

  Background: Deregulated purine metabolism is critical for fast-growing tumor cells by providing nucleotide building blocks and cofactors. Importantly, purine antimetabolites belong to the earliest developed anticancer drugs and are still prescribed in clinics today. However, these antimetabolites can inhibit non-tumor cells and cause undesired side effects. As liver has the highest concentration of purines, it makes liver cancer a good model to study important nodes of dysregulated purine metabolism for better patient selection and precisive cancer treatment.Methods: By using a training dataset from TCGA, we investigated the differentially expressed genes (DEG) of purine metabolism pathway (hsa00230) in hepatocellular carcinoma (HCC) and determined their clinical correlations to patient survival. A prognosis model was established by Lasso-penalized Cox regression analysis, and then validated through multiple examinations including Cox regression analysis, stratified analysis, and nomogram using another ICGC test dataset. We next treated HCC cells using chemical drugs of the key enzymes in vitro to determine targetable candidates in HCC.
Results: The DEG analysis found 43 up-regulated and 2 down-regulated genes in the purine metabolism pathway. Among them, 10 were markedly associated with HCC patient survival. A prognostic correlation model including five genes (PPAT, DCK, ATIC, IMPDH1, RRM2) was established and then validated using the ICGC test dataset. Multivariate Cox regression analysis found that both prognostic risk model (HR = 4.703 or 3.977) and TNM stage (HR = 2.303 or 2.957) independently predicted HCC patient survival in the two datasets respectively. The up-regulations of the five genes were further validated by comparing between 10 pairs of HCC tissues and neighboring non-tumor tissues. In vitro cellular experiments further confirmed that inhibition of IMPDH1 significantly repressed HCC cell proliferation.
Conclusion: In summary, this study suggests that purine metabolism is deregulated in HCC. The prognostic gene correlation model based on the five purine metabolic genes may be useful in predicting HCC prognosis and patient selection. Moreover, the deregulated genes are targetable by specific inhibitors.

Keywords:  bioinformatics; biomarker; hepatocellular carcinoma; prognosis risk model; purine metabolism

DOI:  https://doi.org/10.3389/fonc.2020.583053
Curr Mol Med. 2021 Feb 01.

Deeper Insight in Metastatic Cancer progression;Epithelial-to-Mesenchymal Transition and Genomic Instability: implications on treatment resistance.

Kenneth Omabe, Sandra Uduituma, David Igwe, Maxwell Omabe.

  Therapy resistance remains the major obstacle to successful cancer treatment. Epithelial-to- mesenchymal transition [EMT], a cellular reprogramming process involved in embryogenesis and organ development and regulated by a number of transcriptional factors [EMT-TFs] such as ZEB1/2, is recognized for its role in tumor progression and metastasis. Recently, a growing body of evidence has implicated EMT in cancer therapy resistance but the actual mechanism that underlie this finding has remained elusive. For example, whether it is, the EMT states in itself or the EMT-TFs that modulates chemo or radio-resistance in cancer is still contentious. Here, we summarise the molecular mechanisms of EMT program and chemotherapeutic resistance in cancer with specific reference to DNA damage response [DDR]. We provide an insight into the molecular interplay that exist between EMT program and DNA repair machinery in cancer and how this interaction influences therapeutic response. We review conflicting studies linking EMT and drug resistance via the DNA damage repair axis. We draw scientific evidence demonstrating how several molecular signalling, including EMT-TFs work in operational harmony to induce EMT and confer stemness properties on the EMT-susceptible cells. We highlight the role of enhanced DNA damage repair system associated with EMT-derived stem cell-like states in promoting therapy resistance and suggest a multi-targeting modality in combating cancer treatment resistance.

Keywords:  Cancer; DNA damage repair.; EMT; Treatment resistance; stemness

DOI:  https://doi.org/10.2174/1566524021666210202114844
Oncogene. 2020 Dec 15.

Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.

Manikandan Periyasamy, Anup K Singh, Carolina Gemma, Raed Farzan, Rebecca C Allsopp, Jacqueline A Shaw, Sara Charmsaz, Leonie S Young, Paula Cunnea, R Charles Coombes, Balázs Győrffy, Lakjaya Buluwela, Simak Ali.

The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.

DOI: https://doi.org/10.1038/s41388-020-01583-7
DNA Repair (Amst). 2021 Jan 21. pii: S1568-7864(21)00003-3. [Epub ahead of print]99 103049

The multifaceted roles of DNA repair and replication proteins in aging and obesity.

Alexandra M D'Amico, Karen M Vasquez.

  Efficient mechanisms for genomic maintenance (i.e., DNA repair and DNA replication) are crucial for cell survival. Aging and obesity can lead to the dysregulation of genomic maintenance proteins/pathways and are significant risk factors for the development of cancer, metabolic disorders, and other genetic diseases. Mutations in genes that code for proteins involved in DNA repair and DNA replication can also exacerbate aging- and obesity-related disorders and lead to the development of progeroid diseases. In this review, we will discuss the roles of various DNA repair and replication proteins in aging and obesity as well as investigate the possible mechanisms by which aging and obesity can lead to the dysregulation of these proteins and pathways.

Keywords:  Aging; DNA repair; DNA replication; Genetic instability; Obesity

DOI:  https://doi.org/10.1016/j.dnarep.2021.103049
Mol Genet Metab Rep. 2021 Mar;26 100709

Co-therapy with S-adenosylmethionine and nicotinamide riboside improves t-cell survival and function in Arts Syndrome (PRPS1 deficiency).

Nina Lenherr, John Christodoulou, John Duley, Doreen Dobritzsch, Lynette Fairbanks, Alexandre N Datta, Isabel Filges, Nicolas Gürtler, Jeroen Roelofsen, André B P van Kuilenburg, Claudia Kemper, Erin E West, Gabor Szinnai, Martina Huemer.

  Arts syndrome or phosphoribosyl-pyrophosphate-synthetase-1 (PRPS1) deficiency is caused by loss-of-function mutations in the PRPS1 gene (Xq22.3). PRPS1 is an initial and essential step for the synthesis of the nucleotides of purines, pyrimidines, and nicotinamide. Classically, affected males present with sensorineural hearing loss, optic atrophy, muscular hypotonia, developmental impairment, and recurrent severe respiratory infections early in life. Treatment of a 3-year old boy with S-adenosylmethionine (SAM) replenished erythrocyte purine nucleotides of adenosine and guanosine, while SAM and nicotinamide riboside co-therapy further improved his clinical phenotype as well as T-cell survival and function.

Keywords:  5-phosphoribosyl-1- pyrophosphate, PRPP; Adenosine triphosphate, ATP; Guanosine triphosphate, GTP; NAD phosphate, NADP; Nicotinamide riboside, NR; PRPP synthase, PRPPS; S-adenosylmethionine, SAM; nicotinamide adenine dinucleotide, NAD; phosphoribosyl-pyrophosphate-synthetase-1, PRPS1

DOI:  https://doi.org/10.1016/j.ymgmr.2021.100709
Int J Mol Sci. 2021 Jan 28. pii: 1274. [Epub ahead of print]22(3):

Development of MTH1-Binding Nucleotide Analogs Based on 7,8-Dihalogenated 7-Deaza-dG Derivatives.

Hui Shi, Ren Ishikawa, Choon Han Heh, Shigeki Sasaki, Yosuke Taniguchi.

  MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.

Keywords:  MTH1; dihalogenated nucleoside derivative; nucleotide analog; oxidized nucleotide

DOI:  https://doi.org/10.3390/ijms22031274
Mol Cell Biol. 2021 Feb 01. pii: MCB.00044-20. [Epub ahead of print]

A conserved histone H3-H4 interface regulates DNA damage tolerance and homologous recombination during the recovery from replication stress.

Masafumi Hayashi, Kenji Keyamura, Asami Yoshida, Mariko Ariyoshi, Genki Akanuma, Takashi Hishida.

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. Here we screened a collection of mutation for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress MMS sensitivity of DDT-deficient cells (hereafter we refer to as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with a decreased MMS-induced mutagenesis relative to wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.

DOI: https://doi.org/10.1128/MCB.00044-20
Mutagenesis. 2021 Feb 05. pii: geab007. [Epub ahead of print]

The Role of DNA Polymerase ζ in Benzo[a]pyrene-induced Mutagenesis in the Mouse Lung.

Yuji Ishii, Shinji Takasu, Petr Grúz, Kenichi Masumura, Kumiko Ogawa, Takehiko Nohmi, Takashi Umemura.

  DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency (MF) was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose-response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

Keywords:   gpt delta; Polymerase zeta; Polζ; benzo[a]pyrene; mismatch extension; translesion synthesis (TLS)

DOI:  https://doi.org/10.1093/mutage/geab007
Nat Commun. 2021 01 27. 12(1): 601

APE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling.

Tung-Chang Liu, Chun-Ting Lin, Kai-Cheng Chang, Kai-Wei Guo, Shuying Wang, Jhih-Wei Chu, Yu-Yuan Hsiao.

The exonuclease activity of Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for processing matched/mismatched terminus in various DNA repair pathways and for removing nucleoside analogs associated with drug resistance. To fill in the gap of structural basis for exonucleolytic cleavage, we determine the APE1-dsDNA complex structures displaying end-binding. As an exonuclease, APE1 does not show base preference but can distinguish dsDNAs with different structural features. Integration with assaying enzyme activity and binding affinity for a variety of substrates reveals for the first time that both endonucleolytic and exonucleolytic cleavage can be understood by an induced space-filling model. Binding dsDNA induces RM (Arg176 and Met269) bridge that defines a long and narrow product pocket for exquisite machinery of substrate selection. Our study paves the way to comprehend end-processing of dsDNA in the cell and the drug resistance relating to APE1.

DOI: https://doi.org/10.1038/s41467-020-20853-2
Nat Commun. 2021 02 02. 12(1): 731

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.

Corey H Yu, Akash Bhattacharya, Mirjana Persaud, Alexander B Taylor, Zhonghua Wang, Angel Bulnes-Ramos, Joella Xu, Anastasia Selyutina, Alicia Martinez-Lopez, Kristin Cano, Borries Demeler, Baek Kim, Stephen C Hardies, Felipe Diaz-Griffero, Dmitri N Ivanov.

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

DOI: https://doi.org/10.1038/s41467-021-21023-8
Mol Cell. 2021 Jan 28. pii: S1097-2765(20)30940-0. [Epub ahead of print]

Damage-induced chromatome dynamics link Ubiquitin ligase and proteasome recruitment to histone loss and efficient DNA repair.

Kiran Challa, Christoph D Schmid, Saho Kitagawa, Anaïs Cheblal, Vytautas Iesmantavicius, Andrew Seeber, Assaf Amitai, Jan Seebacher, Michael H Hauer, Kenji Shimada, Susan M Gasser.

  Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.

Keywords:  ▪▪▪

DOI:  https://doi.org/10.1016/j.molcel.2020.12.021
J Biol Chem. 2020 Apr 24. pii: S0021-9258(17)50288-4. [Epub ahead of print]295(17): 5564-5576

Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase.

Parminder Kaur, Matthew J Longley, Hai Pan, Wendy Wang, Preston Countryman, Hong Wang, William C Copeland.

  Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA-binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Keywords:  DNA binding protein; DNA helicase; DNA replication; Twinkle helicase; atomic force microscopy (AFM); mitochondrial DNA (mtDNA); mitochondrial disease; single-molecule biophysics; ssDNA-binding protein (SSB); structural dynamics

DOI:  https://doi.org/10.1074/jbc.RA120.012795
Cell Death Differ. 2021 Feb 02.

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Gwenola Manic, Martina Musella, Francesca Corradi, Antonella Sistigu, Sara Vitale, Sara Soliman Abdel Rehim, Luca Mattiello, Eva Malacaria, Claudia Galassi, Michele Signore, Matteo Pallocca, Stefano Scalera, Frauke Goeman, Francesca De Nicola, Andrea Guarracino, Rosa Pennisi, Fabrizio Antonangeli, Francesca Sperati, Marta Baiocchi, Mauro Biffoni, Maurizio Fanciulli, Marcello Maugeri-Saccà, Annapaola Franchitto, Pietro Pichierri, Ruggero De Maria, Ilio Vitale.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

DOI: https://doi.org/10.1038/s41418-020-00733-4
Molecules. 2021 Feb 02. pii: 771. [Epub ahead of print]26(3):

Design, Synthesis and Biological Evaluation of New Pyrimidine Derivatives as Anticancer Agents.

Valentina Noemi Madia, Alice Nicolai, Antonella Messore, Alessandro De Leo, Davide Ialongo, Valeria Tudino, Francesco Saccoliti, Daniela De Vita, Luigi Scipione, Marco Artico, Samanta Taurone, Ludovica Taglieri, Roberto Di Santo, Susanna Scarpa, Roberta Costi.

  BACKGROUND: Anticancer drug resistance is a challenging phenomenon of growing concern which arises from alteration in drug targets. Despite the fast speed of new chemotherapeutic agent design, the increasing prevalence of this phenomenon requires further research and treatment development. Recently, we reported a new aminopyrimidine compound-namely RDS 344-as a potential innovative anticancer agent.METHODS: Herein, we report the design, synthesis, and anti-proliferative activity of new aminopyrimidine derivatives structurally related to RDS 3442 obtained by carrying out substitutions at position 6 of the pyrimidine core and/or on the 2-aniline ring of our hit. The ability to inhibit cell proliferation was evaluated on different types of tumors, glioblastoma, triple-negative breast cancer, oral squamous cell carcinomas and colon cancer plus on human dermal fibroblasts chosen as control of normal cells.
RESULTS: The most interesting compound was the N-benzyl counterpart of RDS 3442, namely 2a, that induced a significant decrease in cell viability in all the tested tumor cell lines, with EC50s ranging from 4 and 8 μM, 4-13 times more active of hit.
CONCLUSIONS: These data suggest a potential role for this class of molecules as promising tool for new approaches in treating cancers of different histotype.

Keywords:  breast cancer; colon carcinoma; glioblastoma multiforme; lung cancer; microwave reactions; pyrimidine

DOI:  https://doi.org/10.3390/molecules26030771
JCI Insight. 2021 Feb 02. pii: 141553. [Epub ahead of print]

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli.

  Limitations in cell proliferation are important for normal function of differentiated tissues, and essential for the safty of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion and determined growth potential, differentiation and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S-phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Keywords:  Beta cells; Cell Biology; Cell cycle; Stem cell transplantation; Stem cells

DOI:  https://doi.org/10.1172/jci.insight.141553
Br J Haematol. 2021 Feb 02.

Expression of the novel tumour suppressor sterile alpha motif and HD domain-containing protein 1 is an independent adverse prognostic factor in classical Hodgkin lymphoma.

Ioanna Xagoraris, Theodoros P Vassilakopoulos, Elias Drakos, Maria K Angelopoulou, Fotios Panitsas, Nikolas Herold, L Jeffrey Medeiros, Xanthoula Giakoumis, Gerassimos A Pangalis, George Z Rassidakis.

  The expression patterns and prognostic significance of sterile alpha motif and HD domain-containing protein 1 (SAMHD1) protein in the neoplastic Hodgkin and Reed Sternberg (HRS) cells of Hodgkin lymphoma (HL) were investigated in a cohort of 154 patients with HL treated with standard regimens. SAMHD1 expression was assessed by immunohistochemistry using diagnostic lymph node biopsies obtained prior to treatment. Using an arbitrary 20% cut-off, SAMHD1 was positive in HRS cells of 48/154 (31·2%) patients. SAMHD1 expression was not associated with clinicopathologic parameters, such as age, gender, stage or histologic subtype. In 125 patients with a median follow-up of 90 months (7-401 months), SAMHD1 expression in HRS cells significantly correlated with inferior freedom from progression (FFP) (P = 0·025), disease-specific survival (DSS) (P = 0·013) and overall survival (OS) (P = 0·01). Importantly, in multivariate models together with disease stage, histology subtype and type of treatment as covariates, SAMHD1 expression retained an independent significant association with unfavourable FFP (P = 0·005) as well as DSS (P = 0·022) and OS (P = 0·018). These findings uncover the significance of a novel, adverse prognostic factor in HL that may have therapeutic implications since SAMHD1 inhibitors are now available for clinical use.

Keywords:  Hodgkin lymphoma; SAMHD1; prognosis

DOI:  https://doi.org/10.1111/bjh.17352