bims-numges 2022-05-29 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒05‒29
twenty-six papers selected by
Sean Rudd
Karolinska Institutet

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.
Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1.
Recent Advances in Understanding the Structures of Translesion Synthesis DNA Polymerases.
A PARylation-phosphorylation cascade promotes TOPBP1 loading and RPA-RAD51 exchange in homologous recombination.
Smc5/6 Complex Promotes Rad3ATR Checkpoint Signaling at the Perturbed Replication Fork through Sumoylation of the RecQ Helicase Rqh1.
Alternative end-joining originates stable chromosome aberrations induced by etoposide during targeted inhibition of DNA-PKcs in ATM-deficient tumor cells.
In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.
AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/Mediator of DNA damage Checkpoint 1 and repressing MIR22HG/miR-22-3p.
USP49 is a novel deubiquitylating enzyme for γ H2AX in DNA double-strand break repair.
Mutation spectrum data for Saccharomyces cerevisiae psf1-1 pol2-M644G mutants.
RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.
Rtt109 promotes nucleosome replacement ahead of the replicating fork.
The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.
Mutational signatures are markers of drug sensitivity of cancer cells.
Orc6 is a component of the replication fork and enables efficient mismatch repair.
RNase H2, mutated in Aicardi-Goutières syndrome, resolves co-transcriptional R-loops to prevent DNA breaks and inflammation.
OGG1 Inhibition Triggers Synthetic Lethality and Enhances The Effect of PARP Inhibitor Olaparib in BRCA1-Deficient TNBC Cells.
S Phase Duration Is Determined by Local Rate and Global Organization of Replication.
DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.
Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.
Synthetic lethality between TP53 and ENDOD1.
Targeted delivery of a colchicine analogue provides synergy with ATR inhibition in cancer cells.
Polymerase ζ Is Involved in Mitochondrial DNA Maintenance Processes in Concert with APE1 Activity.
Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.
3,3'-Diindolylmethane Enhances Fluorouracil Sensitivity via Inhibition of Pyrimidine Metabolism in Colorectal Cancer.
DDX41 is required for cGAS-STING activation against DNA virus infection.

Cell Death Dis. 2022 May 27. 13(5): 503

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.

María José Peña-Gómez, Paula Moreno-Gordillo, Milda Narmontė, Clara B García-Calderón, Audronė Rukšėnaitė, Saulius Klimašauskas, Iván V Rosado.

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2'-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2'-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2'-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1-/- cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.

DOI: https://doi.org/10.1038/s41419-022-04952-0
Nat Commun. 2022 May 24. 13(1): 2876

Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1.

Tyler M Weaver, Timothy H Click, Thu H Khoang, M Todd Washington, Pratul K Agarwal, Bret D Freudenthal.

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1.

DOI: https://doi.org/10.1038/s41467-022-30577-0
Genes (Basel). 2022 May 20. pii: 915. [Epub ahead of print]13(5):

Recent Advances in Understanding the Structures of Translesion Synthesis DNA Polymerases.

Justin A Ling, Zach Frevert, M Todd Washington.

  DNA damage in the template strand causes replication forks to stall because replicative DNA polymerases are unable to efficiently incorporate nucleotides opposite template DNA lesions. To overcome these replication blocks, cells are equipped with multiple translesion synthesis polymerases that have evolved specifically to incorporate nucleotides opposite DNA lesions. Over the past two decades, X-ray crystallography has provided a wealth of information about the structures and mechanisms of translesion synthesis polymerases. This approach, however, has been limited to ground state structures of these polymerases bound to DNA and nucleotide substrates. Three recent methodological developments have extended our understanding of the structures and mechanisms of these polymerases. These include time-lapse X-ray crystallography, which allows one to identify novel reaction intermediates; full-ensemble hybrid methods, which allow one to examine the conformational flexibility of the intrinsically disordered regions of proteins; and cryo-electron microscopy, which allows one to determine the high-resolution structures of larger protein complexes. In this article, we will discuss how these three methodological developments have added to our understanding of the structures and mechanisms of translesion synthesis polymerases.

Keywords:  DNA damage; DNA polymerases; DNA repair; DNA replication; genome stability; mutagenesis

DOI:  https://doi.org/10.3390/genes13050915
Mol Cell. 2022 May 13. pii: S1097-2765(22)00394-X. [Epub ahead of print]

A PARylation-phosphorylation cascade promotes TOPBP1 loading and RPA-RAD51 exchange in homologous recombination.

Jiao Zhao, Shanshan Tian, Qiushi Guo, Kaiwen Bao, Guohui Yu, Xiaodan Wang, Xilin Shen, Jieyou Zhang, Jiaxin Chen, Ying Yang, Ling Liu, Xiangchun Li, Jihui Hao, Na Yang, Zhe Liu, Ding Ai, Jie Yang, Yi Zhu, Zhi Yao, Shuai Ma, Kai Zhang, Lei Shi.

  The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.

Keywords:  DNA double-strand break; DSB; HR repair; RPA-RAD51 exchange; TOPBP1 loading; genome stability; homologous recombination

DOI:  https://doi.org/10.1016/j.molcel.2022.04.031
Mol Cell Biol. 2022 May 25. e0004522

Smc5/6 Complex Promotes Rad3ATR Checkpoint Signaling at the Perturbed Replication Fork through Sumoylation of the RecQ Helicase Rqh1.

Saman Khan, Nafees Ahamad, Sankhadip Bhadra, Zheng Xu, Yong-Jie Xu.

  Smc5/6, like cohesin and condensin, is a structural maintenance of chromosomes complex crucial for genome stability. Unlike cohesin and condensin, Smc5/6 carries an essential Nse2 subunit with SUMO E3 ligase activity. While screening for new DNA replication checkpoint mutants in fission yeast, we have identified two previously uncharacterized mutants in Smc5/6. Characterization of the mutants and a series of previously reported Smc5/6 mutants uncovered that sumoylation of the RecQ helicase Rqh1 by Nse2 facilitates the checkpoint signaling at the replication fork. We found that mutations that eliminate the sumoylation sites or the helicase activity of Rqh1 compromised the checkpoint signaling similar to a nse2 mutant lacking the ligase activity. Surprisingly, introducing a sumoylation site mutation to a helicase-inactive rqh1 mutant promoted cell survival under stress. These findings, together with other genetic data, support a mechanism that sumoylation of Rqh1 by Smc5/6-Nse2 recruits Rqh1 or modulates its helicase activity at the fork to facilitate the checkpoint signaling. Since the Smc5/6 complex, Rqh1, and the replication checkpoint are conserved in eukaryotes, a similar checkpoint mechanism may be operating in human cells.

Keywords:  ATR; Cds1; Chk1; DNA damage checkpoint; DNA replication checkpoint; Nse2; Rad3; Rqh1; Smc5/6; genome integrity; sumoylation

DOI:  https://doi.org/10.1128/mcb.00045-22
Chromosome Res. 2022 May 23.

Alternative end-joining originates stable chromosome aberrations induced by etoposide during targeted inhibition of DNA-PKcs in ATM-deficient tumor cells.

Marcelo de Campos Nebel, Micaela Palmitelli, Josefina Pérez Maturo, Marcela González-Cid.

  ATM and DNA-PKcs coordinate the DNA damage response at multiple levels following the exposure to chemotherapy. The Topoisomerase II poison etoposide (ETO) is an effective chemotherapeutic agent that induces DNA double-strand breaks (DSB), but it is responsible from the chromosomal rearrangements frequently found in therapy-related secondary tumors. Targeted inhibition of DNA-PKcs in ATM-defective tumors combined with radio- or chemotherapy has been proposed as relevant therapies. Here, we explored the DNA repair mechanisms and the genetic consequences of targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumor cells after exposure to ETO. We demonstrated that chemical inhibition of DNA-PKcs followed by treatment with ETO resulted in the accumulation of chromatid breaks and decreased mitotic index in both A-T cells and ATM-knocked-down (ATMkd) tumor cells. The HR repair process in DNA-PKcs-inhibited ATMkd cells amplified the RAD51 foci number, with no correlated increase in sister chromatid exchanges. The analysis of post-mitotic DNA lesions presented an augmented number of persistent unresolved DSB, without alterations in the cell cycle progression. Long-term examination of chromosome aberrations revealed a strikingly high number of chromatid and chromosome exchanges. By using genetic and pharmacological abrogation of PARP-1, we demonstrated that alternative end-joining (alt-EJ) repair pathway is responsible for those chromosome abnormalities generated by limiting c-NHEJ activities during directed inhibition of DNA-PKcs in ATM-deficient cells. Targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumors stimulates the DSB repair by alt-EJ, which is liable for the origin of cells carrying stable chromosome aberrations that may eventually restrict the therapeutic strategy.

Keywords:  ATM-deficient human cells; DNA and chromosome damages; DNA-PKcs inhibition; cell cycle; double-strand break repair; etoposide

DOI:  https://doi.org/10.1007/s10577-022-09700-w
J Biol Chem. 2022 May 20. pii: S0021-9258(22)00495-1. [Epub ahead of print] 102055

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Cameron Bryan, Xiaoying Wei, Zhishuo Wang, Kun Yang.

  Apurinic/apyrimidinic (AP, or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: 1) 5'-DNA strand incision of the DPC by endonuclease IV (Endo IV); 2-4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and 5) strand ligation by a ligase. We further demonstrated that Endo IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

Keywords:  DNA damage; DNA polymerase I; DNA repair; DNA-protein cross-link; abasic site; base excision repair; endonuclease IV

DOI:  https://doi.org/10.1016/j.jbc.2022.102055
Mol Cancer. 2022 May 27. 21(1): 120

AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/Mediator of DNA damage Checkpoint 1 and repressing MIR22HG/miR-22-3p.

Jinjiang Chou, Markus Kaller, Stephanie Jaeckel, Matjaz Rokavec, Heiko Hermeking.

  BACKGROUND: AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells.METHODS: A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, β-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity.
RESULTS: Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner.
CONCLUSIONS: In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.

Keywords:  AP4; Chemo-resistance; Colorectal cancer; DNA damage; DNA repair; Homologous recombination; MDC1; MIR22HG; c-MYC; miR-22-3p

DOI:  https://doi.org/10.1186/s12943-022-01581-1
Gene. 2022 May 19. pii: S0378-1119(22)00418-8. [Epub ahead of print]833 146599

USP49 is a novel deubiquitylating enzyme for γ H2AX in DNA double-strand break repair.

Misaki Matsui, Shoki Kajita, Yuina Tsuchiya, Wakana Torii, Shiori Tamekuni, Ryotaro Nishi.

  DNA double-strand break (DSB) that is one of the most serious DNA lesions is mainly repaired by two mutually exclusive pathways, homologous recombination and non-homologous end-joining. Proper choice of DSB repair pathway, in which recruitment of 53BP1 to chromatin around DSB sites plays a pivotal role, is crucial for maintaining genome integrity. Ubiquitylations of histone H2A and H2AX on Lys15 are prerequisite for 53BP1 loading onto chromatin. Although ubiquitylation mechanism of H2A and H2AX had been extensively studied, mechanism regulating deubiquitylation of γH2AX that is a phosphorylated form of H2AX remains elusive. Here, we identified USP49 as a novel deubiquitylating enzyme targeting DSB-induced γH2AX ubiquitylation. Over-expressed USP49 suppressed ubiquitylation of γH2AX in an enzymatic activity-dependent manner. Catalytic dead mutant of USP49 interacted and colocalized with γH2AX. Consequently, over-expression of USP49 inhibited the DSB-induced foci formation of 53BP1 and resulted in higher cell sensitivity to DSB-inducing drug treatment. Furthermore, endogenous USP49 protein was degraded via the proteasome upon DSB induction, indicating the importance of modulating USP49 protein level for γH2AX deubiquitylation. Consistent with our cell-based data, kidney renal clear cell carcinoma patients with higher expression of USP49 showed poor survival rate in comparison to the patients with unaltered USP49 expression. In conclusion, these data suggest that fine tuning of protein level of USP49 and USP49-mediated deubiquitylation of γH2AX are important for genome integrity.

Keywords:  53BP1; Cancer; DNA double-strand break repair; Deubiquitylating enzyme; H2AX; Ubiquitylation

DOI:  https://doi.org/10.1016/j.gene.2022.146599
Data Brief. 2022 Jun;42 108223

Mutation spectrum data for Saccharomyces cerevisiae psf1-1 pol2-M644G mutants.

Michal Dmowski, Karolina Makiela-Dzbenska, Malgorzata Jedrychowska, Milena Denkiewicz-Kruk, Iwona J Fijalkowska.

  DNA replication in Saccharomyces cerevisiae and other eukaryotes is performed mainly by polymerase epsilon (Pol ε) on the leading strand and polymerase delta (Pol δ) on the lagging strand. Using a mutant form of a DNA polymerase enables tracking its signature in the replicated DNA. Here, we used the pol2-M644G allele encoding the catalytic subunit of Pol ε to analyse its contribution to DNA replication in yeast with the psf1-1 allele of an essential gene encoding a subunit of the GINS complex. GINS is involved in the recruitment of Pol ε, the major leading strand replicase. Thus, its defective functioning can affect the involvement of Pol ε in DNA replication. Together with Cdc45 and Mcm2-7, GINS forms the CMG helicase complex. Our DNA sequencing data enable the observation of changes in the mutational spectra in the URA3 reporter gene cloned in two orientations regarding the nearest ARS. The data presented in this article support the study "Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex" [1].

Keywords:  CMG (Cdc45 Mcm2-7 GINS); DNA polymerase epsilon (Pol ε); DNA replication fidelity; Genome stability; Replication fork

DOI:  https://doi.org/10.1016/j.dib.2022.108223
J Clin Invest. 2022 May 26. pii: e145660. [Epub ahead of print]

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.

Jonathan Shintaku, Wolfgang M Pernice, Wafaa Eyaid, Jeevan B Gc, Zuben P Brown, Marti Juanola-Falgarona, Javier Torres-Torronteras, Ewen W Sommerville, Debby Mei Hellebrekers, Emma L Blakely, Alan Donaldson, Ingrid Mbh van de Laar, Cheng-Shiun Leu, Ramon Marti, Joachim Frank, Kurenai Tanji, David A Koolen, Richard J Rodenburg, Patrick F Chinnery, H J M Smeets, Gráinne S Gorman, Penelope E Bonnen, Robert W Taylor, Michio Hirano.

  Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders due to impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report five probands from four families who presented with ptosis and ophthalmoplegia, plus other manifestations and multiple mtDNA deletions in muscle. We identified three RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and two homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Keywords:  Genetic diseases; Genetics; Mitochondria; Molecular pathology

DOI:  https://doi.org/10.1172/JCI145660
Genome Res. 2022 May 24. pii: gr.276674.122. [Epub ahead of print]

Rtt109 promotes nucleosome replacement ahead of the replicating fork.

Felix Jonas, Gilad Yaakov, Naama Barkai.

DNA replication perturbs chromatin by triggering the eviction, replacement and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a genomically encoded sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing slow synchronous replication in nucleotide limiting conditions. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S phase-induced acetyltransferase, stabilizes nucleosomes behind the fork, but promotes H3 replacement ahead of the fork. Increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56, but rather results from Vps75-dependent Rtt109 activity towards the H3 N-terminus. Our results suggest that, at least under nucleotide limiting conditions, selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, likely reflecting the distinct challenges for genome stability at these different regions.

DOI: https://doi.org/10.1101/gr.276674.122
Nat Commun. 2022 May 25. 13(1): 2915

The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.

Almutasem Saleh, Yasunori Noguchi, Ricardo Aramayo, Marina E Ivanova, Kathryn M Stevens, Alex Montoya, S Sunidhi, Nicolas Lopez Carranza, Marcin J Skwark, Christian Speck.

The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.

DOI: https://doi.org/10.1038/s41467-022-30576-1
Nat Commun. 2022 May 25. 13(1): 2926

Mutational signatures are markers of drug sensitivity of cancer cells.

Jurica Levatić, Marina Salvadores, Francisco Fuster-Tormo, Fran Supek.

Genomic analyses have revealed mutational footprints associated with DNA maintenance gone awry, or with mutagen exposures. Because cancer therapeutics often target DNA synthesis or repair, we asked if mutational signatures make useful markers of drug sensitivity. We detect mutational signatures in cancer cell line exomes (where matched healthy tissues are not available) by adjusting for the confounding germline mutation spectra across ancestries. We identify robust associations between various mutational signatures and drug activity across cancer cell lines; these are as numerous as associations with established genetic markers such as driver gene alterations. Signatures of prior exposures to DNA damaging agents - including chemotherapy - tend to associate with drug resistance, while signatures of deficiencies in DNA repair tend to predict sensitivity towards particular therapeutics. Replication analyses across independent drug and CRISPR genetic screening data sets reveal hundreds of robust associations, which are provided as a resource for drug repurposing guided by mutational signature markers.

DOI: https://doi.org/10.1038/s41467-022-30582-3
Proc Natl Acad Sci U S A. 2022 May 31. 119(22): e2121406119

Orc6 is a component of the replication fork and enables efficient mismatch repair.

Yo-Chuen Lin, Dazhen Liu, Arindam Chakraborty, Lyudmila Y Kadyrova, You Jin Song, Qinyu Hao, Jaba Mitra, Rosaline Y C Hsu, Mariam K Arif, Sneha Adusumilli, Ting-Wei Liao, Taekjip Ha, Farid A Kadyrov, Kannanganattu V Prasanth, Supriya G Prasanth.

  Significance Origin recognition complex (ORC) is required for the initiation of DNA replication. Unlike other ORC components, the role of human Orc6 in replication remains to be resolved. We identified an unexpected role for hOrc6, which is to promote S-phase progression after prereplication complex assembly and DNA damage response. Orc6 localizes at the replication fork, is an accessory factor of the mismatch repair complex, and plays a fundamental role in genome surveillance during S phase.

Keywords:  ATR; DNA damage; Orc6; mismatch repair; replication

DOI:  https://doi.org/10.1073/pnas.2121406119
Nat Commun. 2022 May 26. 13(1): 2961

RNase H2, mutated in Aicardi-Goutières syndrome, resolves co-transcriptional R-loops to prevent DNA breaks and inflammation.

Agnese Cristini, Michael Tellier, Flavia Constantinescu, Clelia Accalai, Laura Oana Albulescu, Robin Heiringhoff, Nicolas Bery, Olivier Sordet, Shona Murphy, Natalia Gromak.

RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.

DOI: https://doi.org/10.1038/s41467-022-30604-0
Front Oncol. 2022 ;12 888810

OGG1 Inhibition Triggers Synthetic Lethality and Enhances The Effect of PARP Inhibitor Olaparib in BRCA1-Deficient TNBC Cells.

Juan Miguel Baquero, Erik Marchena-Perea, Rocío Mirabet, Raúl Torres-Ruiz, Carmen Blanco-Aparicio, Sandra Rodríguez-Perales, Thomas Helleday, Carlos Benítez-Buelga, Javier Benítez, Ana Osorio.

  Background: PARP1 plays a critical role in the base excision repair (BER) pathway, and PARP1 inhibition leads to specific cell death, through a synthetic lethal interaction, in the context of BRCA1/2 deficiency. To date, up to five different PARP inhibitors (PARPi), have been approved, nevertheless, the acquisition of resistance to PARPi is common and there is increasing interest in enhancing responses and expand their use to other tumour types.Methods: We hypothesized that other BER members could be additional synthetic lethal partners with mutated BRCA genes. To test this, we decided to evaluate the glycosylase OGG1 as a potential candidate, by treating BRCA1 proficient and deficient breast cancer cells with PARPi olaparib and the OGG1 inhibitor TH5478.
Results: Knocking out BRCA1 in triple-negative breast cancer cell lines causes hypersensitivity to the OGG1 inhibitor TH5487. Besides, TH5487 enhances the sensitivity to the PARP inhibitor olaparib, especially in the context of BRCA1 deficiency, reflecting an additive interaction.
Discussion: These results provide the first evidence that OGG1 inhibition is a promising new synthetic lethality strategy in BRCA1-deficient cells, and could lead to a new framework for the treatment of hereditary breast and ovarian cancer.

Keywords:  BRCA1; OGG1 inhibitor; PARP1 inhibitor; synthetic lethality; triple negative breast cancer

DOI:  https://doi.org/10.3389/fonc.2022.888810
Biology (Basel). 2022 May 07. pii: 718. [Epub ahead of print]11(5):

S Phase Duration Is Determined by Local Rate and Global Organization of Replication.

Avraham Greenberg, Itamar Simon.

  The duration of the cell cycle has been extensively studied and a wide degree of variability exists between cells, tissues and organisms. However, the duration of S phase has often been neglected, due to the false assumption that S phase duration is relatively constant. In this paper, we describe the methodologies to measure S phase duration, summarize the existing knowledge about its variability and discuss the key factors that control it. The local rate of replication (LRR), which is a combination of fork rate (FR) and inter-origin distance (IOD), has a limited influence on S phase duration, partially due to the compensation between FR and IOD. On the other hand, the organization of the replication program, specifically the amount of replication domains that fire simultaneously and the degree of overlap between the firing of distinct replication timing domains, is the main determinant of S phase duration. We use these principles to explain the variation in S phase length in different tissues and conditions.

Keywords:  DNA replication; S phase; cell cycle; fork rate; inter-origin distance; replication timing

DOI:  https://doi.org/10.3390/biology11050718
Biochemistry. 2022 May 24.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

Christopher O Dusek, Radha Charan Dash, Kerry S McPherson, Jackson T Calhoun, Irina Bezsonova, Dmitry M Korzhnev, M Kyle Hadden.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.

DOI: https://doi.org/10.1021/acs.biochem.2c00027
Sci Immunol. 2022 May 27. 7(71): eabh4271

Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.

Michael D Claiborne, Srona Sengupta, Liang Zhao, Matthew L Arwood, Im-Meng Sun, Jiayu Wen, Elizabeth A Thompson, Marisa Mitchell-Flack, Marikki Laiho, Jonathan D Powell.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously activated CD8+ T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously activated CD8+ T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels but diminished available pre-rRNA (ribosomal RNA), the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously activated CD8+ T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

DOI: https://doi.org/10.1126/sciimmunol.abh4271
Nat Commun. 2022 May 23. 13(1): 2861

Synthetic lethality between TP53 and ENDOD1.

Zizhi Tang, Ming Zeng, Xiaojun Wang, Chang Guo, Peng Yue, Xiaohu Zhang, Huiqiang Lou, Jun Chen, Dezhi Mu, Daochun Kong, Antony M Carr, Cong Liu.

The atypical nuclease ENDOD1 functions with cGAS-STING in innate immunity. Here we identify a previously uncharacterized ENDOD1 function in DNA repair. ENDOD1 is enriched in the nucleus following H2O2 treatment and ENDOD1-/- cells show increased PARP chromatin-association. Loss of ENDOD1 function is synthetic lethal with homologous recombination defects, with affected cells accumulating DNA double strand breaks. Remarkably, we also uncover an additional synthetic lethality between ENDOD1 and p53. ENDOD1 depletion in TP53 mutated tumour cells, or p53 depletion in ENDOD1-/- cells, results in rapid single stranded DNA accumulation and cell death. Because TP53 is mutated in ~50% of tumours, ENDOD1 has potential as a wide-spectrum target for synthetic lethal treatments. To support this we demonstrate that systemic knockdown of mouse EndoD1 is well tolerated and whole-animal siRNA against human ENDOD1 restrains TP53 mutated tumour progression in xenograft models. These data identify ENDOD1 as a potential cancer-specific target for SL drug discovery.

DOI: https://doi.org/10.1038/s41467-022-30311-w
Biochem Pharmacol. 2022 May 19. pii: S0006-2952(22)00189-7. [Epub ahead of print] 115095

Targeted delivery of a colchicine analogue provides synergy with ATR inhibition in cancer cells.

Francis M Barnieh, Goreti Ribeiro Morais, Herbie Garland, Paul M Loadman, Robert A Falconer.

  Despite significant preclinical promise as anticancer agents, vascular-disrupting agents have yet to fulfil their clinical potential due to systemic toxicities. ICT2588 is a tumour-selective MT1-MMP-targeted prodrug of azademethylcolchicine, ICT2552. We investigate activation of ICT2588 and subsequent release of ICT2552 in tumour cells, and examine its ability to induce G2/M cell cycle arrest. We also explore synergism between ICT2588 and ATR inhibition, since colchicine, in addition to its vascular-disrupting properties, is known to induce G2/M arrest, DNA damage, and trigger apoptosis. Several ATR inhibitors are currently undergoing clinical evaluation. The cellular activation of ICT2588 was observed to correlate with MT1-MMP expression, with selective release of ICT2552 not compromised by cellular uptake and prodrug activation mechanisms. ICT2588 induced G2/M arrest, and triggered apoptosis in MT1-MMP-expressing cells, but not in cells lacking MT1-MMP expression, while ICT2552 itself induced G2/M arrest and triggered apoptosis in both cell lines. Interestingly, we uncovered that the intracellular release and accumulation dynamics of ICT2552 subsequent to prodrug activation provided synergism with an ATR inhibitor in a way not observed with direct administration of ICT2552. These findings have important potential implications for clinical combinations of ICT2588 and DNA repair inhibitors.

Keywords:  ATR; AZD6738; ICT2588; MT1-MMP; colchicine

DOI:  https://doi.org/10.1016/j.bcp.2022.115095
Genes (Basel). 2022 May 13. pii: 879. [Epub ahead of print]13(5):

Polymerase ζ Is Involved in Mitochondrial DNA Maintenance Processes in Concert with APE1 Activity.

Heike Katrin Schreier, Rahel Stefanie Wiehe, Miria Ricchetti, Lisa Wiesmüller.

  Mitochondrial DNA (mtDNA) damaged by reactive oxygen species (ROS) triggers so far poorly understood processes of mtDNA maintenance that are coordinated by a complex interplay among DNA repair, DNA degradation, and DNA replication. This study was designed to identify the proteins involved in mtDNA maintenance by applying a special long-range PCR, reflecting mtDNA integrity in the minor arc. A siRNA screening of literature-based candidates was performed under conditions of enforced oxidative phosphorylation revealing the functional group of polymerases and therein polymerase ζ (POLZ) as top hits. Thus, POLZ knockdown caused mtDNA accumulation, which required the activity of the base excision repair (BER) nuclease APE1, and was followed by compensatory mtDNA replication determined by the single-cell mitochondrial in situ hybridization protocol (mTRIP). Quenching reactive oxygen species (ROS) in mitochondria unveiled an additional, ROS-independent involvement of POLZ in the formation of a typical deletion in the minor arc region. Together with data demonstrating the localization of POLZ in mitochondria, we suggest that POLZ plays a significant role in mtDNA turnover, particularly under conditions of oxidative stress.

Keywords:  base excision repair; mitochondrial DNA degradation; oxidative damage; polymerase ζ

DOI:  https://doi.org/10.3390/genes13050879
Int J Mol Sci. 2022 May 20. pii: 5701. [Epub ahead of print]23(10):

Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.

Nadia Vezzio-Vié, Marie-Alice Kong-Hap, Eve Combès, Augusto Faria Andrade, Maguy Del Rio, Philippe Pasero, Charles Theillet, Céline Gongora, Philippe Pourquier.

  The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.

Keywords:  ATR inhibitor; DNA damage response; DNA repair; anticancer drugs; biomarkers; imaging cytometry; oxaliplatin

DOI:  https://doi.org/10.3390/ijms23105701
Metabolites. 2022 Apr 30. pii: 410. [Epub ahead of print]12(5):

3,3'-Diindolylmethane Enhances Fluorouracil Sensitivity via Inhibition of Pyrimidine Metabolism in Colorectal Cancer.

Jieping Zhang, Shaomin Zou, Yijing Zhang, Ziqing Yang, Wencong Wang, Manqi Meng, Junyan Feng, Peng Zhang, Lishi Xiao, Mong-Hong Lee, Lekun Fang.

  Chemoresistance limits treatment outcomes in colorectal cancer (CRC) patients. A dimeric metabolite of indole-3-carbinol, 3,3'-diindolylmethane (DIM) is abundant in cruciferous vegetables and has shown anticancer efficacy. The role of DIM in regulating chemosensitivity in CRC remains unknown. In this study, we demonstrated that DIM treatment inhibits the malignant progression of CRC. RNA sequencing indicated that pyrimidine synthesis genes are attenuated by DIM treatment. Stable 13C-labeled glucose tracing revealed that DIM inhibits de novo pyrimidine biosynthesis in CRC. DIM increases 5-FU cytotoxicity in CRC via regulation of the expression of pyrimidine metabolism-related genes. DIM synergizes with 5-FU to enhance its inhibitory effects on CRC both in vivo and in vitro. Our results suggest that DIM improves the therapeutic outcomes of FU-based chemotherapy in CRCs by inhibiting pyrimidine metabolism, identifying a new strategy for clinical therapy.

Keywords:  3,3′-Diindolylmethane; chemosensitivity; colorectal cancer; pyrimidine metabolism

DOI:  https://doi.org/10.3390/metabo12050410
Cell Rep. 2022 May 24. pii: S2211-1247(22)00629-5. [Epub ahead of print]39(8): 110856

DDX41 is required for cGAS-STING activation against DNA virus infection.

Ravi Shankar Singh, Venkatasubramanian Vidhyasagar, Shizhuo Yang, Ananna Bhadra Arna, Manisha Yadav, Aanchal Aggarwal, Alexya N Aguilera, Satoru Shinriki, Kalpana Kalyanasundaram Bhanumathy, Kannupriya Pandey, Aizhang Xu, Noreen Rapin, Mark Bosch, John DeCoteau, Jim Xiang, Franco J Vizeacoumar, Yan Zhou, Vikram Misra, Hirotaka Matsui, Susan R Ross, Yuliang Wu.

  Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

Keywords:  AML; CP: Immunology; CP: Molecular biology; DDX41; DNA virus; MDS; STING; acute myeloid leukemia; annealing; cGAS; myelodysplastic syndromes; unwinding

DOI:  https://doi.org/10.1016/j.celrep.2022.110856