bims-numges 2021-06-20 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–06–20
37 papers selected by
Sean Rudd, Karolinska Institutet

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks.
Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex.
mTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer.
Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer.
ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication.
The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation.
ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability.
CIP2A interacts with TopBP1 and drives basal-like breast cancer tumorigenesis.
Post-translational modification of factors involved in homologous recombination.
Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA.
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.
BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.
DNA Polymerase λ Promotes Error-free Replication through Watson-Crick Impairing N1-methyl-deoxyadenosine Adduct in Conjunction with DNA Polymerase ζ.
Cytosine base modifications regulate DNA duplex stability and metabolism.
Targeting Pyrimidine Metabolism in the Era of Precision Cancer Medicine.
Hypoxia-induced SETX links replication stress with the unfolded protein response.
Phase 1 Combination Study of the CHK1 inhibitor prexasertib, and the PARP inhibitor olaparib, in high-grade serous ovarian cancer and other solid tumors.
TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells.
Histone dynamics during DNA replication stress.
Combination of chemotherapy with BRAF inhibitors results in effective eradication of malignant melanoma by preventing ATM-dependent DNA repair.
Shining light on single-strand lesions caused by the chemotherapy drug bleomycin.
RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts.
Two mechanisms of chromosome fragility at replication-termination sites in bacteria.
Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes.
Inhibition of Pim-2 kinase by LT-171-861 promotes DNA damage and exhibits enhanced lethal effects with PARP inhibitor in multiple myeloma.
Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity.
Repair of DNA double-strand breaks in RNAPI- and RNAPII-transcribed loci.
SIRT7: a sentinel of genome stability.
Structural basis for the coiled-coil architecture of human CtIP.
A robust photoluminescence screening assay identifies uracil-DNA glycosylase inhibitors against prostate cancer.
Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.
Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.
Protocol for analysis of G2/M DNA synthesis in human cells.
Targeting nucleotide metabolism as the nexus of viral infections, cancer, and the immune response.
Cytoplasmic RRM1 activation as an acute response to gemcitabine treatment is involved in drug resistance of pancreatic cancer cells.
Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.

Nat Commun. 2021 06 17. 12(1): 3636

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

Diana Zatreanu, Helen M R Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert A Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall M B Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luned M Badder, Daniela Novo, Eleanor G Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Chris Bot, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Katherine Ewings, Wojciech Krajewski, Ellen MacDonald, Hollie McCarron, Leon Pang, Chris Pedder, Laurent Rigoreau, Martin Swarbrick, Ed Wheatley, Simon Willis, Ai Ching Wong, Andre Nussenzweig, Marcel Tijsterman, Andrew Tutt, Simon J Boulton, Geoff S Higgins, Stephen J Pettitt, Graeme C M Smith, Christopher J Lord.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

DOI: https://doi.org/10.1038/s41467-021-23463-8
EMBO J. 2021 Jun 15. e106355

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks.

Daniel González-Acosta, Elena Blanco-Romero, Patricia Ubieto-Capella, Karun Mutreja, Samuel Míguez, Susana Llanos, Fernando García, Javier Muñoz, Luis Blanco, Massimo Lopes, Juan Méndez.

  DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Keywords:  ICL repair; ICL traverse; PrimPol; RPA; interstrand crosslink

DOI:  https://doi.org/10.15252/embj.2020106355
Sci Adv. 2021 May;pii: eabf2278. [Epub ahead of print]7(21):

Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex.

Robin van Schendel, Ron Romeijn, Helena Buijs, Marcel Tijsterman.

During genome duplication, the replication fork encounters a plethora of obstacles in the form of damaged bases, DNA-cross-linked proteins, and secondary structures. How cells protect DNA integrity at sites of stalled replication is currently unknown. Here, by engineering "primase deserts" into the Caenorhabditis elegans genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss. We next identify the pol α-primase complex to limit deletion mutagenesis, a conclusion substantiated by whole-genome analysis of animals carrying mutated POLA2/DIV-1. We subsequently identify a new role for the 9-1-1 checkpoint clamp in protecting Okazaki fragments from resection by EXO1. Together, our results provide a mechanistic model for controlling the fate of replication intermediates at sites of stalled replication.

DOI: https://doi.org/10.1126/sciadv.abf2278
Neoplasia. 2021 Jun 11. pii: S1476-5586(21)00032-4. [Epub ahead of print]23(7): 643-652

mTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer.

Ling Tian, Congcong Chen, Yanguan Guo, Fan Zhang, Jinye Mi, Qi Feng, Shengbin Lin, Naite Xi, Jiaxin Tian, Li Yu, Yan Chen, Mingrong Cao, Caiyong Lai, Jun Fan, Yongchang Zhang, Guo Chen.

  Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells.

Keywords:  DNA replication stress; Gemcitabine; Ribonucleotide reductase; mTORC2

DOI:  https://doi.org/10.1016/j.neo.2021.05.007
Nucleic Acids Res. 2021 Jun 18. pii: gkab492. [Epub ahead of print]

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer.

Teruya Nakamura, Kohtaro Okabe, Shogo Hirayama, Mami Chirifu, Shinji Ikemizu, Hiroshi Morioka, Yusaku Nakabeppu, Yuriko Yamagata.

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.

DOI: https://doi.org/10.1093/nar/gkab492
Sci Adv. 2021 Jun;pii: eabd9208. [Epub ahead of print]7(25):

ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication.

Dejie Wang, Jian Ma, Maria Victoria Botuyan, Gaofeng Cui, Yuqian Yan, Donglin Ding, Yingke Zhou, Eugene W Krueger, Jiang Pei, Xiaosheng Wu, Liguo Wang, Huadong Pei, Mark A McNiven, Dingwei Ye, Georges Mer, Haojie Huang.

53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase-catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

DOI: https://doi.org/10.1126/sciadv.abd9208
Nat Commun. 2021 06 10. 12(1): 3520

The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation.

Xiaoping Xu, Kai Ni, Yafeng He, Jianke Ren, Chongkui Sun, Yie Liu, Mirit I Aladjem, Sandra Burkett, Richard Finney, Xia Ding, Shyam K Sharan, Kathrin Muegge.

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.

DOI: https://doi.org/10.1038/s41467-021-23809-2
iScience. 2021 Jun 25. 24(6): 102534

ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability.

Shibin Xu, Xingxuan Wu, Peipei Wang, Sheng-Li Cao, Bin Peng, Xingzhi Xu.

  DNA double-strand break (DSB) repair by homologous recombination (HR) is essential for ensuring genome stability. Abnormal spindle-like microcephaly-associated (ASPM) gene encodes a spindle protein that is commonly implicated in primary microcephaly. We found that ASPM is recruited to sites of DNA damage in a PARP2-dependent manner. ASPM interacts with BRCA1 and its E3 ligase HERC2, preventing HERC2 from accessing to BRCA1 and ensuring BRCA1 stability. Inhibition of ASPM expression promotes HERC2-mediated BRCA1 degradation, compromises HR repair efficiency and chromosome stability, and sensitizes cancer cells to ionizing radiation. Moreover, we observed a synergistic effect between ASPM and PARP inhibition in killing cancer cells. This research has uncovered a novel function for ASPM in facilitating HR-mediated repair of DSBs by ensuring BRCA1 stability. ASPM might constitute a promising target for synthetic lethality-based cancer therapy.

Keywords:  Cell biology; Functional aspects of cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2021.102534
Cancer Res. 2021 Jun 18. pii: canres.3651.2020. [Epub ahead of print]

CIP2A interacts with TopBP1 and drives basal-like breast cancer tumorigenesis.

Anni Laine, Srikar G Nagelli, Caroline Farrington, Umar Butt, Anna N Cvrljevic, Julia P Vainonen, Femke M Feringa, Tove J Grönroos, Prson Gautam, Sofia Khan, Harri Sihto, Xi Qiao, Karolina Pavic, Denise C Connolly, Pauliina Kronqvist, Laura L Elo, Jochen Maurer, Krister Wennerberg, Rene H Medema, Heikki Joensuu, Emilia Peuhu, Karin de Visser, Goutham Narla, Jukka Westermarck.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA-damage-induced initiation of mouse BLBC-like mammary tumors and for survival of homologous recombination defective (HRD) BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA-damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small molecule reactivators of PP2A (SMAPs) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibit growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA-damage induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3651
DNA Repair (Amst). 2021 Jun 07. pii: S1568-7864(21)00070-7. [Epub ahead of print]104 103114

Post-translational modification of factors involved in homologous recombination.

Bilge Argunhan, Hiroshi Iwasaki, Hideo Tsubouchi.

  DNA is the molecule that stores the chemical instructions necessary for life and its stability is therefore of the utmost importance. Despite this, DNA is damaged by both exogenous and endogenous factors at an alarming frequency. The most severe type of DNA damage is a double-strand break (DSB), in which a scission occurs in both strands of the double helix, effectively dividing a single normal chromosome into two pathological chromosomes. Homologous recombination (HR) is a universal DSB repair mechanism that solves this problem by identifying another region of the genome that shares high sequence similarity with the DSB site and using it as a template for repair. Rad51 possess the enzymatic activity that is essential for this repair but several auxiliary factors are required for Rad51 to fulfil its function. It is becoming increasingly clear that many HR factors are subjected to post-translational modification. Here, we review what is known about how these modifications affect HR. We first focus on cases where there is experimental evidence to support a function for the modification, then discuss speculative cases where a function can be inferred. Finally, we contemplate why such modifications might be necessary.

Keywords:  Acetylation; DNA repair; Genome stability; Phosphorylation; Rad51; Recombination; SUMOylation; Ubiquitination; Ubiquitylation

DOI:  https://doi.org/10.1016/j.dnarep.2021.103114
Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2106393118. [Epub ahead of print]118(25):

Rtt105 promotes high-fidelity DNA replication and repair by regulating the single-stranded DNA-binding factor RPA.

Xuejie Wang, Yang Dong, Xiaocong Zhao, Jinbao Li, Jordan Lee, Zhenxin Yan, Shuangshuang Yang, Wenqiang Wu, Ximiao Hou, Guangxue Liu, Yueyue Zhang, Lun Song, Gang Cai, Qing Li, Grzegorz Ira, Xinghua Zhang, Xuefeng Chen.

  Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.

Keywords:  RPA; Rtt105; genome stability; homologous recombination; replication fidelity

DOI:  https://doi.org/10.1073/pnas.2106393118
EMBO J. 2021 Jun 15. 40(12): e106393

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Nasim A Begum, Farazul Haque, Andre Stanlie, Afzal Husain, Samiran Mondal, Mikiyo Nakata, Takako Taniguchi, Hisaaki Taniguchi, Tasuku Honjo.

  Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

Keywords:  CSR; H2A.Z; NHEJ; Phf5a; genomic instability

DOI:  https://doi.org/10.15252/embj.2020106393
Nat Commun. 2021 06 10. 12(1): 3542

BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.

Jekaterina Vohhodina, Liana J Goehring, Ben Liu, Qing Kong, Vladimir V Botchkarev, Mai Huynh, Zhiqi Liu, Fieda O Abderazzaq, Allison P Clark, Scott B Ficarro, Jarrod A Marto, Elodie Hatchi, David M Livingston.

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.

DOI: https://doi.org/10.1038/s41467-021-23716-6
J Biol Chem. 2021 Jun 10. pii: S0021-9258(21)00668-2. [Epub ahead of print] 100868

DNA Polymerase λ Promotes Error-free Replication through Watson-Crick Impairing N1-methyl-deoxyadenosine Adduct in Conjunction with DNA Polymerase ζ.

Jung-Hoon Yoon, Debashree Basu, Jayati Roy Choudhury, Satya Prakash, Louise Prakash.

  In a previous study we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting a nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ - which normally uses W-C base pairing for DNA synthesis - in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.

Keywords:  DNA polymerase ζ; DNA polymerase λ; Hoogsteen base pairing; N1-methyl-deoxyadenosine; Translesion synthesis; error-free TLS by DNA polymerase λ

DOI:  https://doi.org/10.1016/j.jbc.2021.100868
Nucleic Acids Res. 2021 Jun 16. pii: gkab509. [Epub ahead of print]

Cytosine base modifications regulate DNA duplex stability and metabolism.

Cathia Rausch, Peng Zhang, Corella S Casas-Delucchi, Julia L Daiß, Christoph Engel, Gideon Coster, Florian D Hastert, Patrick Weber, M Cristina Cardoso.

DNA base modifications diversify the genome and are essential players in development. Yet, their influence on DNA physical properties and the ensuing effects on genome metabolism are poorly understood. Here, we focus on the interplay of cytosine modifications and DNA processes. We show by a combination of in vitro reactions with well-defined protein compositions and conditions, and in vivo experiments within the complex networks of the cell that cytosine methylation stabilizes the DNA helix, increasing its melting temperature and reducing DNA helicase and RNA/DNA polymerase speed. Oxidation of methylated cytosine, however, reverts the duplex stabilizing and genome metabolic effects to the level of unmodified cytosine. We detect this effect with DNA replication and transcription proteins originating from different species, ranging from prokaryotic and viral to the eukaryotic yeast and mammalian proteins. Accordingly, lack of cytosine methylation increases replication fork speed by enhancing DNA helicase unwinding speed in cells. We further validate that this cannot simply be explained by altered global DNA decondensation, changes in histone marks or chromatin structure and accessibility. We propose that the variegated deposition of cytosine modifications along the genome regulates DNA helix stability, thereby providing an elementary mechanism for local fine-tuning of DNA metabolism.

DOI: https://doi.org/10.1093/nar/gkab509
Front Oncol. 2021 ;11 684961

Targeting Pyrimidine Metabolism in the Era of Precision Cancer Medicine.

Wanyan Wang, Jiayan Cui, Hui Ma, Weiqiang Lu, Jin Huang.

  Metabolic rewiring is considered as a primary feature of cancer. Malignant cells reprogram metabolism pathway in response to various intrinsic and extrinsic drawback to fuel cell survival and growth. Among the complex metabolic pathways, pyrimidine biosynthesis is conserved in all living organism and is necessary to maintain cellular fundamental function (i.e. DNA and RNA biosynthesis). A wealth of evidence has demonstrated that dysfunction of pyrimidine metabolism is closely related to cancer progression and numerous drugs targeting pyrimidine metabolism have been approved for multiple types of cancer. However, the non-negligible side effects and limited efficacy warrants a better strategy for negating pyrimidine metabolism in cancer. In recent years, increased studies have evidenced the interplay of oncogenic signaling and pyrimidine synthesis in tumorigenesis. Here, we review the recent conceptual advances on pyrimidine metabolism, especially dihydroorotate dehydrogenase (DHODH), in the framework of precision oncology medicine and prospect how this would guide the development of new drug precisely targeting the pyrimidine metabolism in cancer.

Keywords:  dihydroorotate dehydrogenase; metabolic reprogram; precision medicine; pyrimidine inhibitor; pyrimidine metabolism

DOI:  https://doi.org/10.3389/fonc.2021.684961
Nat Commun. 2021 06 17. 12(1): 3686

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Shaliny Ramachandran, Tiffany S Ma, Jon Griffin, Natalie Ng, Iosifina P Foskolou, Ming-Shih Hwang, Pedro Victori, Wei-Chen Cheng, Francesca M Buffa, Katarzyna B Leszczynska, Sherif F El-Khamisy, Natalia Gromak, Ester M Hammond.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.

DOI: https://doi.org/10.1038/s41467-021-24066-z
Clin Cancer Res. 2021 Jun 15. pii: clincanres.1279.2021. [Epub ahead of print]

Phase 1 Combination Study of the CHK1 inhibitor prexasertib, and the PARP inhibitor olaparib, in high-grade serous ovarian cancer and other solid tumors.

Khanh Do, Bose S Kochupurakkal, Sarah Kelland, Adrienne de Jonge, Jennifer Hedglin, Allison Powers, Nicholas Quinn, Courtney Gannon, Loan Vuong, Kalindi Parmar, Jean-Bernard Lazaro, Alan D D'Andrea, Geoffrey I Shapiro.

PURPOSE: Checkpoint kinase 1 (CHK1) plays a central role in the response to replication stress through modulation of cell cycle checkpoints and homologous recombination (HR) repair. In BRCA-deficient cancers with de novo or acquired PARP inhibitor resistance, the addition of the CHK1 inhibitor prexasertib to the PARP inhibitor olaparib compromises replication fork stability, as well as HR proficiency, allowing for sensitization to PARP inhibition.
EXPERIMENTAL DESIGN: This study followed a 3+3 design with a 7-day lead-in of olaparib alone, followed by 28-day cycles with prexasertib administered on days 1 and 15 in combination with an attenuated dose of olaparib on days 1-5 and 15-19. Pharmacokinetic blood samples were collected after olaparib alone and following combination therapy. Patients (pts) enrolled to the expansion phase of the study underwent paired tumor biopsies for pharmacodynamic (PD) assessments.
RESULTS: Twenty-nine pts were treated. DLTs included Gr 3 neutropenia and Gr 3 febrile neutropenia. The MTD/RP2D was prexasertib at 70mg/m2 IV with olaparib at 100 mg PO BID. Most common treatment-related AEs included leukopenia (83%), neutropenia (86%), thrombocytopenia (66%) and anemia (72%). Four of 18 pts with BRCA1-mutant, PARP inhibitor-resistant, high-grade serous ovarian cancer (HGSOC) achieved PRs. Paired tumor biopsies demonstrated reduction in RAD51 foci and increased expression of g-H2AX, pKAP1 and pRPA after combination exposure.
CONCLUSIONS: Prexasertib combined with olaparib has preliminary clinical activity in pts with BRCA-mutant HGSOC who have previously progressed on a PARP inhibitor. PD analyses show that prexasertib compromises HR with evidence of induction of DNA damage and replication stress.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-1279
Nat Commun. 2021 Jun 18. 12(1): 3760

TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells.

Bruno Silva, Rajika Arora, Silvia Bione, Claus M Azzalin.

Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.

DOI: https://doi.org/10.1038/s41467-021-24097-6
J Biomed Sci. 2021 Jun 19. 28(1): 48

Histone dynamics during DNA replication stress.

Chia-Ling Hsu, Shin Yen Chong, Chia-Yeh Lin, Cheng-Fu Kao.

  Accurate and complete replication of the genome is essential not only for genome stability but also for cell viability. However, cells face constant threats to the replication process, such as spontaneous DNA modifications and DNA lesions from endogenous and external sources. Any obstacle that slows down replication forks or perturbs replication dynamics is generally considered to be a form of replication stress, and the past decade has seen numerous advances in our understanding of how cells respond to and resolve such challenges. Furthermore, recent studies have also uncovered links between defects in replication stress responses and genome instability or various diseases, such as cancer. Because replication stress takes place in the context of chromatin, histone dynamics play key roles in modulating fork progression and replication stress responses. Here, we summarize the current understanding of histone dynamics in replication stress, highlighting recent advances in the characterization of fork-protective mechanisms.

Keywords:  Genome instability; Histone dynamics; Histone modifications; Histone variants; Replication stress

DOI:  https://doi.org/10.1186/s12929-021-00743-5
Oncogene. 2021 Jun 17.

Combination of chemotherapy with BRAF inhibitors results in effective eradication of malignant melanoma by preventing ATM-dependent DNA repair.

Josune Alonso-Marañón, Alberto Villanueva, Josep Maria Piulats, María Martínez-Iniesta, Laura Solé, Juan Martín-Liberal, Sonia Segura, Ramon M Pujol, Mar Iglesias, Anna Bigas, Fernando Gallardo, Lluís Espinosa.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy in advanced stages. Chemotherapy has not demonstrated its efficacy in MM and current treatment for tumors carrying the most frequent BRAFV600E mutation consists of BRAF inhibitors alone or in combination with MAPK pathway inhibitors. We previously found that BRAF inhibition prevents activation of the DNA-damage repair (DDR) pathway in colorectal cancer thus potentiating the effect of chemotherapy. We now show that different chemotherapy agents inflict DNA damage in MM cells, which is efficiently repaired, associated with activation of the ATM-dependent DDR machinery. Pharmacologic inhibition of BRAF impairs ATM and DDR activation in these cells, leading to sustained DNA damage. Combination treatments involving DNA-damaging agents and BRAF inhibitors increase tumor cell death in vitro and in vivo, and impede MM regrowth after treatment cessation. We propose to reconsider the use of chemotherapy in combination with BRAF inhibitors for MM treatment.

DOI: https://doi.org/10.1038/s41388-021-01879-2
DNA Repair (Amst). 2021 Jun 10. pii: S1568-7864(21)00109-9. [Epub ahead of print]105 103153

Shining light on single-strand lesions caused by the chemotherapy drug bleomycin.

Vandana Singh, Pegah Johansson, Yii-Lih Lin, Ola Hammarsten, Fredrik Westerlund.

  Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.

Keywords:  BER inhibition; DNA damage; Damage mechanism of drugs; Fluorescence microscopy; Personalizing chemotherapy; Single molecule imaging; Single-strand breaks

DOI:  https://doi.org/10.1016/j.dnarep.2021.103153
Nucleic Acids Res. 2021 Jun 16. pii: gkab479. [Epub ahead of print]

RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts.

Wenjie Wang, Kuan Li, Zhuo Yang, Quancan Hou, Wei W Zhao, Qianwen Sun.

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.

DOI: https://doi.org/10.1093/nar/gkab479
Sci Adv. 2021 Jun;pii: eabe2846. [Epub ahead of print]7(25):

Two mechanisms of chromosome fragility at replication-termination sites in bacteria.

Qian Mei, Devon M Fitzgerald, Jingjing Liu, Jun Xia, John P Pribis, Yin Zhai, Ralf B Nehring, Jacob Paiano, Heyuan Li, Andre Nussenzweig, P J Hastings, Susan M Rosenberg.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

DOI: https://doi.org/10.1126/sciadv.abe2846
Front Cell Dev Biol. 2021 ;9 672510

Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes.

Maksym Shyian, David Shore.

  During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome via an evolutionarily conserved Fork Pausing/Protection Complex (FPC). Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.

Keywords:  MTC (Mrc1-Tof1-Csm3); fork pausing complex; replication fork; replication fork barrier (RFB); replication fork slowdown; topoisomerase I (Top1); topoisomerase II (Top2)

DOI:  https://doi.org/10.3389/fcell.2021.672510
Biochem Pharmacol. 2021 Jun 07. pii: S0006-2952(21)00261-6. [Epub ahead of print]190 114648

Inhibition of Pim-2 kinase by LT-171-861 promotes DNA damage and exhibits enhanced lethal effects with PARP inhibitor in multiple myeloma.

Cen Zhao, Dawei Yang, Yuchen Ye, Zhenzhong Chen, Tifan Sun, Jiawei Zhao, Kai Zhao, Na Lu.

  Multiple myeloma (MM) is a malignancy of antibody-producing plasma cells with genomic instability and genetic abnormality as its two hallmarks. Therefore, DNA damage is pervasive in MM cells, which indicates irregular DNA damage response (DDR) pathway. In this study, we demonstrated that LT-171-861, a multiple kinase inhibitor, could inhibit proliferation and induce apoptosis in MM cells. LT-171-861 promoted DDR pathway and triggered DNA damage through impeding the process of homologous recombination in double strand breaks, rather than directly elevating ROS level in MM cells. Mechanism research revealed that Pim2 inhibition was responsible for LT-171-861-indcued DNA damage and cell apoptosis. LT-171-861 mainly suppressed Pim2 kinase activity and reduced the expression of its phosphorylated substrates, such as 4EBP1 and BAD. Moreover, Olaparib, a PARP inhibitor, could enhance the antitumor effect of LT-171-861 in suppressing tumor growth in MM xenografted nude mice. Taken together, our results demonstrated that LT-171-861 showed a promising therapeutic potential for MM and had an additional lethal effect with PARP inhibitors.

Keywords:  Apoptosis; DNA damage response; Multiple myeloma; Pim2

DOI:  https://doi.org/10.1016/j.bcp.2021.114648
Transl Oncol. 2021 Jun 09. pii: S1936-5233(21)00139-X. [Epub ahead of print]14(9): 101147

Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity.

Jennifer Garbarino, Jillian Eckroate, Ranjini K Sundaram, Ryan B Jensen, Ranjit S Bindra.

  Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention.

Keywords:  ATRX; DNA damage response; Glioma; IDH1 R132H; PARP inhibitor

DOI:  https://doi.org/10.1016/j.tranon.2021.101147
DNA Repair (Amst). 2021 May 21. pii: S1568-7864(21)00095-1. [Epub ahead of print]104 103139

Repair of DNA double-strand breaks in RNAPI- and RNAPII-transcribed loci.

E Lesage, T Clouaire, G Legube.

  DNA double-strand breaks (DSBs) are toxic lesions triggered not only by environmental sources, but also by a large number of physiological processes. Of importance, endogenous DSBs frequently occur in genomic loci that are transcriptionally active. Recent work suggests that DSBs occurring in transcribed loci are handled by specific pathway(s) that entail local transcriptional repression, chromatin signaling, the involvement of RNA species and DSB mobility. In this Graphical Review we provide an updated view of the "Transcription-Coupled DSB Repair" (TC-DSBR) pathway(s) that are mounted at DSBs occurring in loci transcribed by RNA Polymerase I (RNAPI) or RNA Polymerase II (RNAPII), highlighting differences and common features, as well as yet unanswered questions.

Keywords:  Genome integrity; RNA polymerase I; RNA polymerase II; Transcription-Coupled DSB Repair

DOI:  https://doi.org/10.1016/j.dnarep.2021.103139
Open Biol. 2021 Jun;11(6): 210047

SIRT7: a sentinel of genome stability.

Ming Tang, Huangqi Tang, Bo Tu, Wei-Guo Zhu.

  SIRT7 is a class III histone deacetylase that belongs to the sirtuin family. The past two decades have seen numerous breakthroughs in terms of understanding SIRT7 biological function. We now know that this enzyme is involved in diverse cellular processes, ranging from gene regulation to genome stability, ageing and tumorigenesis. Genomic instability is one hallmark of cancer and ageing; it occurs as a result of excessive DNA damage. To counteract such instability, cells have evolved a sophisticated regulated DNA damage response mechanism that restores normal gene function. SIRT7 seems to have a critical role in this response, and it is recruited to sites of DNA damage where it recruits downstream repair factors and directs chromatin regulation. In this review, we provide an overview of the role of SIRT7 in DNA repair and maintaining genome stability. We pay particular attention to the implications of SIRT7 function in cancer and ageing.

Keywords:  DNA repair; SIRT7; ageing; cancer; genome stability

DOI:  https://doi.org/10.1098/rsob.210047
Open Biol. 2021 Jun;11(6): 210060

Structural basis for the coiled-coil architecture of human CtIP.

C R Morton, N J Rzechorzek, J D Maman, M Kuramochi, H Sekiguchi, R Rambo, Y C Sasaki, O R Davies, L Pellegrini.

  The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.

Keywords:  DNA repair; coiled-coil structure; human CtIP

DOI:  https://doi.org/10.1098/rsob.210060
Chem Sci. 2020 Jan 10. 11(7): 1750-1760

A robust photoluminescence screening assay identifies uracil-DNA glycosylase inhibitors against prostate cancer.

Guodong Li, Stuart Adam Henry, Hao Liu, Tian-Shu Kang, Sang-Cuo Nao, Yichao Zhao, Chun Wu, Jianwen Jin, Jia-Tong Zhang, Chung-Hang Leung, Philip Wai Hong Chan, Dik-Lung Ma.

Many cancers have developed resistance to 5-FU, due to removal by the enzyme uracil-DNA glycosylase (UDG), a type of base excision repair enzyme (BER) that can excise uracil and 5-fluorouracil (5-FU) from DNA. However, the development of UDG inhibitor screening methods, especially for the rapid and efficient screening of natural product/natural product-like compounds, is still limited so far. We developed herein a robust time-resolved photoluminescence method for screening UDG inhibitors, which could significantly improve sensitivity over the screening method based on the conventional steady-state spectroscopy, reducing the substantial fluorescence background interference. As a proof-of-concept, two potential UDG inhibitors were identified from a database of natural products and approved drugs. Co-treatment of these two compounds with 5-FU showed synergistic cytotoxicity, providing the basis for treating drug-resistant cancers. Overall, this method provides an avenue for the rapid screening of small molecule regulators of other BER enzyme activities that can avoid false negatives arising from the background fluorescence.

DOI: https://doi.org/10.1039/c9sc05623h
Nat Commun. 2021 Jun 18. 12(1): 3759

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.

Miao Guo, Yina Wang, Yuyue Tang, Zijing Chen, Jinfeng Hou, Jingli Dai, Yudong Wang, Liangyan Wang, Hong Xu, Bing Tian, Yuejin Hua, Ye Zhao.

Pol μ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol μ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol μ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol μ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol μ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.

DOI: https://doi.org/10.1038/s41467-021-24096-7
PLoS One. 2021 ;16(6): e0253170

Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.

Handan Sevim, Haydar Çelik, Levent Düşünceli, Ceyda S Ceyhan, Anna Molotkova, Kay Nakazawa, Garrett T Graham, Jeffrey R Petro, Jeffrey A Toretsky, Aykut Üren.

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.

DOI: https://doi.org/10.1371/journal.pone.0253170
STAR Protoc. 2021 Jun 18. 2(2): 100570

Protocol for analysis of G2/M DNA synthesis in human cells.

Jianming Wang, Marco Saponaro.

  G2/M DNA synthesis (G-MiDS) can be observed in one in five G2/M cells in unperturbed conditions by immunofluorescence microscopy. However, little is known of the genomic sites undergoing G-MiDS. Here, we describe a protocol which allows enriching for G2/M cells and investigating the sites of G-MiDS using BrdU-seq. This method can also be used to study the role of DNA replication or transcription-associated factors in affecting G-MiDS levels in different cell lines. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Keywords:  Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Molecular Biology; Sequencing

DOI:  https://doi.org/10.1016/j.xpro.2021.100570
Sci Adv. 2021 May;pii: eabg6165. [Epub ahead of print]7(21):

Targeting nucleotide metabolism as the nexus of viral infections, cancer, and the immune response.

Yarden Ariav, James H Ch'ng, Heather R Christofk, Noga Ron-Harel, Ayelet Erez.

Virus-infected cells and cancers share metabolic commonalities that stem from their insatiable need to replicate while evading the host immune system. These similarities include hijacking signaling mechanisms that induce metabolic rewiring in the host to up-regulate nucleotide metabolism and, in parallel, suppress the immune response. In both cancer and viral infections, the host immune cells and, specifically, lymphocytes augment nucleotide synthesis to support their own proliferation and effector functions. Consequently, established treatment modalities targeting nucleotide metabolism against cancers and virally infected cells may result in restricted immune response. Encouragingly, following the introduction of immunotherapy against cancers, multiple studies improved our understanding for improving antigen presentation to the immune system. We propose here that understanding the immune consequences of targeting nucleotide metabolism against cancers may be harnessed to optimize therapy against viral infections.

DOI: https://doi.org/10.1126/sciadv.abg6165
PLoS One. 2021 ;16(6): e0252917

Cytoplasmic RRM1 activation as an acute response to gemcitabine treatment is involved in drug resistance of pancreatic cancer cells.

Tomotaka Kato, Hiroaki Ono, Mikiya Fujii, Keiichi Akahoshi, Toshiro Ogura, Kosuke Ogawa, Daisuke Ban, Atsushi Kudo, Shinji Tanaka, Minoru Tanabe.

BACKGROUND: RRM1 is functionally associated with DNA replication and DNA damage repair. However, the biological activity of RRM1 in pancreatic cancer remains undetermined.
METHODS: To determine relationships between RRM1 expression and the prognosis of pancreatic cancer, and to explore RRM1 function in cancer biology, we investigated RRM1 expression levels in 121 pancreatic cancer patients by immunohistochemical staining and performed in vitro experiments to analyze the functional consequences of RRM1 expression.
RESULTS: Patients with high RRM1 expression had significantly poorer clinical outcomes (overall survival; p = 0.006, disease-free survival; p = 0.0491). In particular, high RRM1 expression was also associated with poorer overall survival on adjuvant chemotherapy (p = 0.008). We found that RRM1 expression was increased 24 hours after exposure to gemcitabine and could be suppressed by histone acetyltransferase inhibition. RRM1 activation in response to gemcitabine exposure was induced mainly in the cytoplasm and cytoplasmic RRM1 activation was related to cancer cell viability. In contrast, cancer cells lacking cytoplasmic RRM1 activation were confirmed to show severe DNA damage. RRM1 inhibition with specific siRNA or hydroxyurea enhanced the cytotoxic effects of gemcitabine for pancreatic cancer cells.
CONCLUSIONS: Cytoplasmic RRM1 activation is involved in biological processes related to drug resistance in response to gemcitabine exposure and could be a potential target for pancreatic cancer treatment.

DOI: https://doi.org/10.1371/journal.pone.0252917
Elife. 2021 Jun 18. pii: e61407. [Epub ahead of print]10

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.

Kerrie-Ann McMahon, David A Stroud, Yann Gambin, Vikas Tillu, Michele Bastiani, Emma Sierecki, Mark E Polinkovsky, Thomas E Hall, Guillermo A Gomez, Yeping Wu, Marie-Odile Parat, Nick Martel, Harriet P Lo, Kum Kum Khanna, Kirill Alexandrov, Roger Daly, Alpha Yap, Michael T Ryan, Robert G Parton.

  Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.

Keywords:  cancer biology; cell biology

DOI:  https://doi.org/10.7554/eLife.61407