bims-numges 2022-02-13 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒02‒13
25 papers selected by
Sean Rudd
Karolinska Institutet

Pathway specific effects of ADSL deficiency on neurodevelopment.
Guanosine primes acute myeloid leukemia for differentiation via guanine nucleotide salvage synthesis.
DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR.
The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins.
Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.
RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.
Facile preparation of model DNA interstrand cross-link repair intermediates using ribonucleotide-containing DNA.
Multiple roles of Pol epsilon in eukaryotic chromosome replication.
Transcription-replication coordination revealed in single live cells.
Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers.
DNA-Damage-Response-Targeting Mitochondria-Activated Multifunctional Prodrug Strategy for Self-defensive Tumor Therapy.
The role of PARP inhibitors in gastrointestinal cancers.
Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.
Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis.
DNA repair, recombination, and damage signaling.
Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair.
Genotoxic aldehydes in the hematopoietic system.
Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.
Disruption of DNA Repair and Survival Pathways through Heat Shock Protein inhibition by Onalespib to Sensitize Malignant Gliomas to Chemoradiation therapy.
Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.
Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer.
The remarkable gymnastics of ORC.
Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.
Mitochondrial DHODH regulates hypoxia-inducible factor 1 expression in OTSCC.
TOPBP1 regulates resistance of gastric cancer to oxaliplatin by promoting transcription of PARP1.

Elife. 2022 Feb 08. pii: e70518. [Epub ahead of print]11

Pathway specific effects of ADSL deficiency on neurodevelopment.

Ilaria Dutto, Julian Gerhards, Antonio Herrera, Olga Souckova, Václava Škopová, Jordann Smak, Alexandra Junza, Oscar Yanes, Cedric Boeckx, Martin D Burkhalter, Marie Zikánová, Sebastian Pons, Melanie Philipp, Jens Lüders, Travis H Stracker.

  Adenylosuccinate Lyase (ADSL) functions in de novo purine biosynthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

Keywords:  cell biology; chicken; developmental biology; human; zebrafish

DOI:  https://doi.org/10.7554/eLife.70518
Am J Cancer Res. 2022 ;12(1): 427-444

Guanosine primes acute myeloid leukemia for differentiation via guanine nucleotide salvage synthesis.

Hanying Wang, Xin He, Zheng Li, Hongchuan Jin, Xian Wang, Ling Li.

Differentiation arrest represents a distinct hallmark of acute myeloid leukemia (AML). Identification of differentiation-induction agents that are effective across various subtypes remains an unmet challenge. GTP biosynthesis is elevated in several types of cancers, considered to support uncontrolled tumor growth. Here we report that GTP overload by supplementation of guanosine, the nucleoside precursor of GTP, poises AML cells for differentiation and growth inhibition. Transcriptome profiling of guanosine-treated AML cells reveals a myeloid differentiation pattern. Importantly, the treatment compromises leukemia progression in AML xenograft models. Mechanistically, GTP overproduction requires sequential metabolic conversions executed by the purine salvage biosynthesis pathway including the involvement of purine nucleoside phosphorylase (PNP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). Taken together, our study offers novel metabolic insights tethering GTP homeostasis to myeloid differentiation and provides an experimental basis for further clinical investigations of guanosine or guanine nucleotides in the treatment of AML patients.

Keywords: Acute myeloid leukemia; HPRT1; PNP; guanosine 5’-triphosphate; myeloid differentiation
DNA Repair (Amst). 2022 Feb 01. pii: S1568-7864(22)00018-0. [Epub ahead of print]111 103289

DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR.

Michelle L Swift, Jane Azizkhan-Clifford.

  The factors involved in DNA damage recognition and repair are tightly regulated to ensure proper repair pathway choice. The mechanism(s) that determines the cell cycle-dependent turnover of these DNA damage repair factors remains unclear. Here, we show that Sp1, which regulates double-strand break (DSB) repair pathway choice through localization of 53BP1, is sumoylated at Lys16 following DNA damage; Sp1 sumoylation is required for its degradation and the removal of both Sp1 and 53BP1 from DSB sites. Induction of DNA DSBs induces Sp1 phosphorylation at DSBs by ATM, which is necessary for the subsequent sumoylation of Sp1. In addition to this damage-induced ATM-dependent phosphorylation and sumoylation, phosphorylation of Sp1 at Ser59 by Cyclin A/cdk2 upon entry into S phase is necessary for recognition, ubiquitination and degradation by the SUMO-targeted E3 ubiquitin ligase, RNF4. Eliminating Sp1 sumoylation by mutation of Sp1 at Lys16 (K16R) precluded removal of both Sp1 and 53BP1 from DSBs in S phase, resulting in decreased BRCA1 recruitment and defective homologous recombination (HR). Like BRCA1 deficient cells, cells expressing Sp1K16R are sensitive to PARP inhibition due to failure to degrade Sp1 and recruit BRCA1 resulting in defective HR that is rescued by knockdown of 53BP1. These results reveal the dynamic regulation of Sp1 and its role in the assembly and disassembly of DNA repair factors at DSBs.

Keywords:  DSB repair; RNF4; Sp1; StUbL; Ubiquitination

DOI:  https://doi.org/10.1016/j.dnarep.2022.103289
Curr Genet. 2022 Feb 11.

The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins.

Jeff Bachant, Elizabeth A Hoffman, Chris Caridi, Constance I Nugent, Wenyi Feng.

  Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress.

Keywords:  DNA polymerase alpha; DNA replication origin firing; Dbf4; MCM; Primase

DOI:  https://doi.org/10.1007/s00294-022-01230-6
Curr Genet. 2022 Feb 12.

Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.

Hovik Gasparayan, Chris Caridi, Jeff Julius, Wenyi Feng, Jeff Bachant, Constance I Nugent.

  Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

Keywords:  DNA replication origin; DNA replication stress; MCM; Rad53; S phase checkpoint; Stn1

DOI:  https://doi.org/10.1007/s00294-022-01228-0
Nucleic Acids Res. 2022 Feb 08. pii: gkac073. [Epub ahead of print]

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

Ayeong So, Elodie Dardillac, Ali Muhammad, Catherine Chailleux, Laura Sesma-Sanz, Sandrine Ragu, Eric Le Cam, Yvan Canitrot, Jean Yves Masson, Pauline Dupaigne, Bernard S Lopez, Josée Guirouilh-Barbat.

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.

DOI: https://doi.org/10.1093/nar/gkac073
DNA Repair (Amst). 2022 Jan 31. pii: S1568-7864(22)00015-5. [Epub ahead of print]111 103286

Facile preparation of model DNA interstrand cross-link repair intermediates using ribonucleotide-containing DNA.

Jin Tang, Feng Tang, Linlin Zhao.

  DNA interstrand cross-links (ICLs) are lesions with a covalent bond formed between DNA strands. ICLs are extremely toxic to cells because they prevent the separation of the two strands, which are necessary for the genetic interpretation of DNA. ICLs are repaired via Fanconi anemia and replication-independent pathways. The formation of so-called unhooked repair intermediates via a dual strand incision flanking the ICL site on one strand is an essential step in nearly all ICL repair pathways. Recently, ICLs derived from endogenous sources, such as those from ubiquitous DNA lesions, abasic (AP) sites, have emerged as an important class of ICLs. Despite the earlier efforts in preparing AP-ICLs in high yield using nucleotide analogs, little information is available for preparing AP-ICL unhooked intermediates with varying lengths of overhangs. In this study, we devise a simple approach to prepare model ICL unhooked intermediates derived from AP sites. We exploited the alkaline lability of ribonucleotides (rNMPs) and the high cross-linking efficiency between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination. We designed chimeric DNA/RNA substrates with rNMPs flanking the cross-linking residue (2-aminopurine) to facilitate subsequent strand cleavage under our optimized conditions. Mass spectrometric analysis and primer extension assays confirmed the structures of ICL substrates. The method is straightforward, requires no synthetic chemistry expertise, and should be broadly accessible to all researchers in the DNA repair community. For step-by-step descriptions of the method, please refer to the companion manuscript in MethodsX.

Keywords:  DNA damage; DNA interstrand cross-links; DNA repair; RNase H; Translesion synthesis (TLS)

DOI:  https://doi.org/10.1016/j.dnarep.2022.103286
Biochem Soc Trans. 2022 Feb 07. pii: BST20210082. [Epub ahead of print]

Multiple roles of Pol epsilon in eukaryotic chromosome replication.

Milos A Cvetkovic, Esther Ortega, Roberto Bellelli, Alessandro Costa.

  Pol epsilon is a tetrameric assembly that plays distinct roles during eukaryotic chromosome replication. It catalyses leading strand DNA synthesis; yet this function is dispensable for viability. Its non-catalytic domains instead play an essential role in the assembly of the active replicative helicase and origin activation, while non-essential histone-fold subunits serve a critical function in parental histone redeposition onto newly synthesised DNA. Furthermore, Pol epsilon plays a structural role in linking the RFC-Ctf18 clamp loader to the replisome, supporting processive DNA synthesis, DNA damage response signalling as well as sister chromatid cohesion. In this minireview, we discuss recent biochemical and structural work that begins to explain various aspects of eukaryotic chromosome replication, with a focus on the multiple roles of Pol epsilon in this process.

Keywords:  CMG; Ctf18; Pol epsilon; chromatin; cryo-EM; replisome

DOI:  https://doi.org/10.1042/BST20210082
Nucleic Acids Res. 2022 Feb 08. pii: gkac069. [Epub ahead of print]

Transcription-replication coordination revealed in single live cells.

Ioannis Tsirkas, Daniel Dovrat, Manikandan Thangaraj, Ineke Brouwer, Amit Cohen, Zohar Paleiov, Michael M Meijler, Tineke Lenstra, Amir Aharoni.

The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.

DOI: https://doi.org/10.1093/nar/gkac069
Am J Cancer Res. 2022 ;12(1): 17-47

Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers.

Arathi Rajan, Geetu R Varghese, Induprabha Yadev, Jaimie Anandan, Neetha R Latha, Dipyaman Patra, Neethu Krishnan, Krithiga Kuppusamy, Arathy V Warrier, Satej Bhushan, Revathy Nadhan, Ram Mohan Ram Kumar, Priya Srinivas.

BRCA1 mutation carriers have a greater risk of developing cancers in hormone-responsive tissues like breasts and ovaries. However, this tissue-specific incidence of BRCA1 related cancers remains elusive. The majority of the BRCA1 mutated breast cancers exhibit typical histopathological features of high-grade tumors, with basal epithelial phenotype, classified as triple-negative molecular subtype and have a higher percentage of DNA damage and chromosomal abnormality. Though there are many studies relating BRCA1 with ER-α (Estrogen receptor-α), it has not been reported whether E2 (Estrogen) -ER-α signaling can modulate the DNA repair activities of BRCA1. The present study analyzes whether deregulation of ER-α signaling, arising as a result of E2/ER-α deficiency, could impact the BRCA1 dependent DDR (DNA Damage Response) pathways, predominantly those of DNA-DSB (Double Strand break) repair and oxidative damage response. We demonstrate that E2/E2-stimulated ER-α can augment BRCA1 mediated high fidelity repairs like HRR (Homologous Recombination Repair) and BER (Base Excision Repair) in breast cancer cells. Conversely, a condition of ER-α deficiency itself or any interruption in ligand-dependent ER-α transactivation resulted in delayed DNA damage repair, leading to persistent activation of γH2AX and retention of unrepaired DNA lesions, thereby triggering tumor progression. ER-α deficiency not only limited the HRR in cells but also facilitated the DSB repair through error prone pathways like NHEJ (Non Homologous End Joining). ER-α deficiency associated persistence of DNA lesions and reduced expression of DDR proteins were validated in human mammary tumors.

Keywords: BRCA1; DNA damage repair; ER-α; double-strand break (DNA-DSB); homologous recombination repair (HRR)
Angew Chem Int Ed Engl. 2022 Feb 08.

DNA-Damage-Response-Targeting Mitochondria-Activated Multifunctional Prodrug Strategy for Self-defensive Tumor Therapy.

Paramesh Jangili, Na Kong, Ji Hyeon Kim, Jun Zhou, Haijun Liu, Xingcai Zhang, Wei Tao, Jong Seung Kim.

  We report a novel multifunctional construct, M1, designed explicitly to target the DNA damage response in cancer cells. M1 contains both a floxuridine (FUDR) and protein phosphatase 2A (PP2A) inhibitor combined with a GSH-sensitive linker. Further conjugation of the triphenylphosphonium moiety allows M1 to undergo specific activation in the mitochondria, where mitochondria-mediated apoptosis is observed. Moreover, M1 has enormous effects on genomic DNA ascribed to FUDR's primary function of impeding DNA/RNA synthesis combined with diminishing PP2A-activated DNA repair pathways. Importantly, mechanistic studies highlight the PP2A obtrusion in FUDR/5-fluorouracil (5-FU) therapy and underscore the importance of its inhibition to harbor therapeutic potential. HCT116 cell xenograft-bearing mice that have a low response rate to 5-FU show a prominent effect with M1, emphasizing the importance of DNA damage response targeting strategies using tumor-specific microenvironment-activatable systems.

Keywords:  DNA damage response; DNA repair; drug delivery; floxuridine; protein phosphatase 2A

DOI:  https://doi.org/10.1002/anie.202117075
Crit Rev Oncol Hematol. 2022 Feb 03. pii: S1040-8428(22)00045-2. [Epub ahead of print]171 103621

The role of PARP inhibitors in gastrointestinal cancers.

Daire Hanna, Neha Chopra, Daniel Hochhauser, Khurum Khan.

  The use of BReast CAncer (BRCA) mutations as biomarkers for sensitivity to DNA damage response (DDR) targeted drugs and platinum agents is well documented in breast and gynaecological cancers. More recently the successful use DDR targeted therapies including poly (ADP-ribose) polymerases (PARP) inhibitors has been shown to extend to other germline and somatic deficiencies within the homologous recombination (HR) pathway (Farmer et al., 2005; Turner et al., 2019; Li and Heyer, 2008). Gastrointestinal (GI) cancers are lagging behind other tumour types when it comes to personalising treatment with targeted therapies. Current methods of identifying PARP-inhibitor sensitivity in gastrointestinal cancers are based on analogies from other cancer types despite there being a lack of uniformity in determining HR status between tumour types. There is an urgent clinical need to better understand the treatment implications of DDR alterations in gastrointestinal cancers. We have reviewed PARP-inhibitor use in pancreatic, gastroesophageal, hepatobiliary and colorectal cancers and explored HRD as a biomarker for sensitivity to PARP-inhibitors.

Keywords:  Colorectal; Gastroesophageal; Gastrointestinal cancer; Hepatobiliary; Homologous recombination deficiency; PARP-inhibitor; Pancreatic

DOI:  https://doi.org/10.1016/j.critrevonc.2022.103621
PLoS Pathog. 2022 Feb;18(2): e1010275

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Changkun Hu, Taylor Bugbee, Dalton Dacus, Rachel Palinski, Nicholas Wallace.

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

DOI: https://doi.org/10.1371/journal.ppat.1010275
PLoS Genet. 2022 Feb 07. 18(2): e1010051

Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis.

Ádám Póti, Bernadett Szikriszt, Judit Zsuzsanna Gervai, Dan Chen, Dávid Szüts.

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase kappa. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10-20% of non-paired substitutions as well, underscoring the importance of the process.

DOI: https://doi.org/10.1371/journal.pgen.1010051
Genetics. 2022 Feb 04. pii: iyab178. [Epub ahead of print]220(2):

DNA repair, recombination, and damage signaling.

Anton Gartner, JoAnne Engebrecht.

  DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.

Keywords:  DNA repair; WormBook; checkpoint signaling; recombination

DOI:  https://doi.org/10.1093/genetics/iyab178
Nat Commun. 2022 Feb 09. 13(1): 760

Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair.

J Ferreira da Silva, G P Oliveira, E A Arasa-Verge, C Kagiou, A Moretton, G Timelthaler, J Jiricny, J I Loizou.

Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. Here, to determine which cellular factors affect PE efficiency, we carry out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2-17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

DOI: https://doi.org/10.1038/s41467-022-28442-1
Blood. 2022 Feb 11. pii: blood.2019004316. [Epub ahead of print]

Genotoxic aldehydes in the hematopoietic system.

Meng Wang, Felix A Dingler, Ketan J Patel.

DNA damage threatens the integrity of hematopoietic stem cells (HSCs) and the production of blood. Over the last 10 years, a wealth of genetic and functional research has shown that simple reactive aldehydes such as formaldehyde are potent genotoxins to HSCs. Mammals have evolved a two-tier protection mechanism against aldehydes, consisting of aldehyde detoxification enzymes (tier one), and the Fanconi Anemia (FA) DNA repair pathway (tier two) to process any aldehyde-induced DNA damage. Loss of either tier of protection in humans results in defective hematopoiesis and predisposition to leukemia. This review will focus on the impact of aldehydes on hematopoiesis, how they cause DNA damage, the sources of endogenous aldehydes and potential novel protective pathways.

DOI: https://doi.org/10.1182/blood.2019004316
Med Oncol. 2022 Feb 12. 39(5): 50

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Ekjot Kaur, Madhura Ketkar, Shilpee Dutt.

  Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Keywords:  ATM; ATR; Glioblastoma; Radioresistant; Recurrence

DOI:  https://doi.org/10.1007/s12032-022-01657-4
Clin Cancer Res. 2022 Feb 09. pii: clincanres.0468.2020. [Epub ahead of print]

Disruption of DNA Repair and Survival Pathways through Heat Shock Protein inhibition by Onalespib to Sensitize Malignant Gliomas to Chemoradiation therapy.

Jihong Xu, Pei-Jung Wu, Tzung-Huei Lai, Pratibha Sharma, Alessandro Canella, Alessandra M Welker, Christine Beattie, Cynthia D Timmers, Frederick F Lang, Naduparambil K Jacob, J Bradley Elder, Russell Lonser, Michelle Easley, Maciej Pietrzak, Deepa Sampath, Vinay K Puduvalli.

PURPOSE: Proficient DNA repair by homologous recombination (HR) facilitates resistance to chemo-radiation in glioma stem cells (GSCs). We evaluated whether compromising HR by targeting HSP90, a molecular chaperone required for the function of key HR proteins, using onalespib, a long-acting, brain-penetrant HSP90 inhibitor, would sensitize high-grade gliomas to chemo-radiation in vitro and in vivo Experimental Design: The ability of onalespib to deplete HR client proteins, impair HR repair capacity, and sensitize GBM to chemo-radiation was evaluated in vitro in GSCs, and in vivo using zebrafish and mouse intracranial glioma xenograft models. The effects of HSP90 inhibition on the transcriptome and cytoplasmic proteins was assessed in GSCs and in ex vivo organotypic human glioma slice cultures.RESULTS: Treatment with onalespib depleted CHK1 and RAD51, two key proteins of the HR pathway, and attenuated HR repair, sensitizing GSCs to the combination of radiation and temozolomide (TMZ). HSP90 inhibition reprogrammed the transcriptome of GSCs and broadly altered expression of cytoplasmic proteins including known and novel client proteins relevant to GSCs. The combination of onalespib with radiation and TMZ extended survival in a zebra fish and a mouse xenograft model of GBM compared to the standard of care (radiation and TMZ) or onalespib with radiation.
CONCLUSIONS: The results of this study demonstrate that targeting HR by HSP90 inhibition sensitizes GSCs to radiation and chemotherapy and extends survival in zebrafish and mouse intracranial models of GBM. These results provide a preclinical rationale for assessment of HSP90 inhibitors in combination with chemoradiation in GBM patients.

DOI: https://doi.org/10.1158/1078-0432.CCR-20-0468
Nature. 2022 Feb 09.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

Genomics England Research Consortium

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

DOI: https://doi.org/10.1038/s41586-022-04403-y
Nat Commun. 2022 Feb 11. 13(1): 835

Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer.

I-M Launonen, N Lyytikäinen, J Casado, E A Anttila, A Szabó, U-M Haltia, C A Jacobson, J R Lin, Z Maliga, B E Howitt, K C Strickland, S Santagata, K Elias, A D D'Andrea, P A Konstantinopoulos, P K Sorger, A Färkkilä.

The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generate spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes. We identify a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we report a prognostic role of a proliferative tumor-cell subpopulation, which associates with enhanced spatial tumor-immune interactions by CD8+ and CD4 + T-cells in the BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the potential to improve immunotherapeutic strategies and patient stratification in HGSC.

DOI: https://doi.org/10.1038/s41467-022-28389-3
Elife. 2022 02 09. pii: e76475. [Epub ahead of print]11

The remarkable gymnastics of ORC.

Bruce Stillman.

  As a cell prepares to divide, a molecular actor known as the Origin Recognition Complex makes intricate ATP-driven movements to recruit proteins required to duplicate DNA.

Keywords:  Cdt1; Mcm2-7; ORC; S. cerevisiae; biochemistry; chemical biology; chromosomes; gene expression; helicase; origin licensing; replication

DOI:  https://doi.org/10.7554/eLife.76475
Elife. 2022 02 08. pii: e68677. [Epub ahead of print]11

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

Catalina Pereira, Gerardo A Arroyo-Martinez, Matthew Z Guo, Michael S Downey, Emma R Kelly, Kathryn J Grive, Shantha K Mahadevaiah, Jennie R Sims, Vitor M Faca, Charlton Tsai, Carl J Schiltz, Niek Wit, Heinz Jacobs, Nathan L Clark, Raimundo Freire, James Turner, Amy M Lyndaker, Miguel A Brieno-Enriquez, Paula E Cohen, Marcus B Smolka, Robert S Weiss.

  DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Keywords:  9-1-1 complex; ATR; DNA break repair; DNA damage response; cell biology; genetics; genomics; meiosis; meiotic silencing; mouse

DOI:  https://doi.org/10.7554/eLife.68677
Am J Cancer Res. 2022 ;12(1): 48-67

Mitochondrial DHODH regulates hypoxia-inducible factor 1 expression in OTSCC.

Wei Gao, Lingyin Hu, Minjuan Zhang, Shuai Liu, Shaowei Xu, Velda Ling-Yu Chow, Jimmy Yu-Wai Chan, Thian-Sze Wong.

Oral tongue squamous cell carcinoma (OTSCC) was one of the most hypoxic tumors with unfavorable outcomes. Hypoxia-inducible factor-1 (HIF-1) signaling was associated with cancer proliferation, lymph node metastasis, angiogenesis and poor prognosis of OTSCC. Dihydroorotate dehydrogenase (DHODH) catalyzed the rate-limiting step in the de novo pyrimidine biosynthesis. The aim of the study was to explore the biological function of DHODH and investigate whether DHODH regulated HIF-1 signaling in OTSCC. Proliferation, migration and anoikis resistance were used to determine the function of DHODH. Western blot and luciferase activity assays were used to determine the regulatory role of DHODH on HIF-1. We found that increased DHODH expression was associated with advanced tumor stage and poorly differentiated tumor in head and neck cancer patients in The Cancer Genome Atlas (TCGA). DHODH enhanced the proliferation and aggressiveness of OTSCC. Moreover, DHODH prompted tumor growth and metastasis in vivo. DHODH promoted transcription, protein stability, and transactivation activity of HIF1A. DHODH-induced HIF1A upregulation in OTSCC can be reversed by reactive oxygen species (ROS) scavenger, indicating that DHODH enhanced HIF1A expression via ROS production. DHODH inhibitor suppressed DHODH-mediated ROS generation and HIF1A upregulation. Targeting DHODH using clinically available inhibitor, atovaquone, might provide a new strategy to treat OTSCC.

Keywords: DHODH; HIF1A; OTSCC; ROS; atovaquone
DNA Repair (Amst). 2022 Feb 01. pii: S1568-7864(22)00007-6. [Epub ahead of print]111 103278

TOPBP1 regulates resistance of gastric cancer to oxaliplatin by promoting transcription of PARP1.

Zejun Fang, Chaoju Gong, Zhuoni Ye, Wei Wang, Minjing Zhu, Yanyan Hu, Zhiguo Liu, Weihua Zhou, Hongzhang Li.

  Gastric cancer (GC) is the third leading cause of cancer-associated mortality worldwide. The platinum derivative oxaliplatin is widely applied in standard GC chemotherapy but recurrence and metastasis are common in advanced GC cases due to intrinsic or induced chemoresistance. Poly(ADP-Ribose) polymerase 1 (PARP1) is an enzyme crucial for repairing DNA damage induced by platinum compounds, which undermines the effectiveness of platinum-based chemotherapy. Data from the current study showed that topoisomerase IIβ binding protein 1 (TOPBP1), an interacting partner of topoisomerase IIβ, is highly expressed in oxaliplatin-resistant GC (OR-GC) cells and promotes PARP1 transcription through direct binding to its proximal promoter region. Furthermore, AKT-mediated phosphorylation of TOPBP1 at Ser1159 was indispensable for inducing PARP1 expression in OR-GC cells. Disruption of the TOPBP1/PARP1 regulatory pathway decreased cell viability and augmented apoptosis of OR-GC cells. The positive correlation between TOPBP1 and PARP1 was confirmed using both the TCGA database and immunohistochemical analysis of GC tissues. In GC patients receiving oxaliplatin treatment, high expression of TOPBP1 or PARP1 was associated with poor prognosis. Our finding that the TOPBP1/PARP1 pathway facilitates acquisition of oxaliplatin resistance uncovers a novel mechanism underlying platinum-based chemotherapy resistance in gastric cancer that may be utilized for developing effective therapeutic strategies.

Keywords:  Gastric cancer (GC); Oxaliplatin resistance; Poly(ADP-ribose) polymerase 1 (PARP1); Topoisomerase IIβ binding protein 1 (TOPBP1); Transcription

DOI:  https://doi.org/10.1016/j.dnarep.2022.103278