Ribonucleotide Incorporation Research Articles

Second generation lethality in RNAseH2a knockout zebrafish.

Removal of ribonucleotides from DNA by RNaseH2 is essential for genome stability, and its impacted function causes the neurodegenerative disease, Aicardi Goutières Syndrome. We have created a zebrafish rnaseh2a mutant to model this process. Surprisingly, RNaseH2a knockouts show little phenotypic abnormality at adulthood in the first generation, unlike mouse knockout models, which are early embryonic lethal. However, the second generation offspring show reduced development, increased ribonucleotide incorporation and upregulation of key inflammatory markers, resulting in both maternal and paternal embryonic lethality. Thus, neither fathers or mothers can generate viable offspring even when crossed to wild-type partners. Despite their survival, rnaseh2a-/- adults show an accumulation of ribonucleotides in both the brain and testes that is not present in early development. Our data suggest that homozygotes possess RNaseH2 independent compensatory mechanisms that are inactive or overwhelmed by the inherited ribonucleotides in their offspring, or that zebrafish have a yet unknown tolerance mechanism. Additionally, we identify ribodysgenesis, the rapid removal of rNMPs and subsequently lethal fragmentation of DNA as responsible for maternal and paternal embryonic lethality.

Constraints on the emergence of RNA through non-templated primer extension with mixtures of potentially prebiotic nucleotides.

The emergence of RNA on the early Earth is likely to have been influenced by chemical and physical processes that acted to filter out various alternative nucleic acids. For example, UV photostability is thought to have favored the survival of the canonical nucleotides. In a recent proposal for the prebiotic synthesis of the building blocks of RNA, ribonucleotides share a common pathway with arabino- and threo-nucleotides. We have therefore investigated non-templated primer extension with 2-aminoimidazole-activated forms of these alternative nucleotides to see if the synthesis of the first oligonucleotides might have been biased in favor of RNA. We show that non-templated primer extension occurs predominantly through 5'-5' imidazolium-bridged dinucleotides, echoing the mechanism of template-directed primer extension. Ribo- and arabino-nucleotides exhibited comparable rates and yields of non-templated primer extension, whereas threo-nucleotides showed lower reactivity. Competition experiments confirmed the bias against the incorporation of threo-nucleotides. The incorporation of an arabino-nucleotide at the end of the primer acts as a chain terminator and blocks subsequent extension. These biases, coupled with potentially selective prebiotic synthesis, and the templated copying that is known to favour the incorporation of ribonucleotides, provide a plausible model for the effective exclusion of arabino- and threo-nucleotides from primordial oligonucleotides.

Strand specificity of ribonucleotide excision repair in Escherichia coli.

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase-DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand.

Fidelity of Ribonucleotide Incorporation by the SARS-CoV-2 Replication Complex

The SARS-CoV-2 coronavirus has caused a global pandemic. Despite the initial success of vaccines at preventing infection, genomic variation has led to the proliferation of variants capable of higher infectivity. Mutations in the SARS-CoV-2 genome are the consequence of replication errors, highlighting the importance of understanding the determinants of SARS-CoV-2 replication fidelity. The RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit for SARS-CoV-2 RNA replication and genome transcription. Here, we report the fidelity of ribonucleotide incorporation by SARS-CoV-2 RdRp (nsp12), along with its co-factors nsp7/nsp8, using steady-state kinetic analysis. Our analysis suggests that in the absence of the proofreading subunit (nsp14), the nsp12/7/8 complex has a surprisingly low base substitution fidelity (10−1–10−3). This is orders of magnitude lower than the fidelity reported for other coronaviruses (10−6–10−7), highlighting the importance of proofreading for faithful SARS-CoV-2 replication. We performed a mutational analysis of all reported SARS-CoV-2 genomes and identified mutations in both nsp12 and nsp14 that appear likely to lower viral replication fidelity through mechanisms that include impairing the nsp14 exonuclease activity or its association with the RdRp. Our observations provide novel insight into the mechanistic basis of replication fidelity in SARS-CoV-2 and the potential effect of nsp12 and nsp14 mutations on replication fidelity, informing the development of future antiviral agents and SARS-CoV-2 vaccines.

Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.

Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.

Detection and Quantitation of DNA Damage on a Genome-wide Scale Using RADAR-seq.

The formation and persistence of DNA damage can impact biological processes such as DNA replication and transcription. To maintain genome stability and integrity, organisms rely on robust DNA damage repair pathways. Techniques to detect and locate DNA damage sites across a genome enable an understanding of the consequences of DNA damage as well as how damage is repaired, which can have key diagnostic and therapeutic implications. Importantly, advancements in technology have enabled the development of high-throughput sequencing-based DNA damage detection methods. These methods require DNA enrichment or amplification steps that limit the ability to quantitate the DNA damage sites. Further, each of these methods is typically tailored to detect only a specific type of damage. RAre DAmage and Repair (RADAR) sequencing is a DNA sequencing workflow that overcomes these limitations and enables detection and quantitation of DNA damage sites in any organism on a genome-wide scale. RADAR-seq works by replacing DNA damage sites with a patch of modified bases that can be directly detected by Pacific Biosciences Single-Molecule Real Time sequencing. Here, we present three protocols that enable detection of thymine dimers and ribonucleotides in bacterial and archaeal genomes. Basic Protocol 1 enables construction of a reference genome required for RADAR-seq analyses. Basic Protocol 2 describes how to locate, quantitate, and compare thymine dimer levels in Escherichia coli exposed to varying amounts of UV light. Basic Protocol 3 describes how to locate, quantitate, and compare ribonucleotide levels in wild-type and ΔRNaseH2 Thermococcus kodakarensis. Importantly, all three protocols provide in-depth steps for data analysis. Together they serve as proof-of-principle experiments that will allow users to adapt the protocols to locate and quantitate a wide variety of DNA damage sites in any organism. © 2022 New England Biolabs. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Constructing a reference genome utilizing SMRT sequencing Basic Protocol 2: Mapping and quantitating genomic thymine dimer formation in untreated versus UV-irradiated E. coli using RADAR-seq Basic Protocol 3: Mapping and quantitating genomic ribonucleotide incorporation in wildtype versus ΔRNaseH2 T. kodakarensis using RADAR-seq.

ATP enhances the error-prone ribonucleotide incorporation by the SARS-CoV-2 RNA polymerase

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved “core” replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.

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

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by 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 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 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 that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Ribodysgenesis: sudden genome instability in the yeast Saccharomyces cerevisiae arising from RNase H2 cleavage at genomic-embedded ribonucleotides.

Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term ‘ribodysgenesis’. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.

Ribonucleotide Incorporation by Eukaryotic B-Family Replicases and Its Implications for Genome Stability.

Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.

DTYMK is essential for genome integrity and neuronal survival

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Ribose-Map: A bioinformatics toolkit for ribonucleotide sequencing experiments

<h2>Abstract</h2> Current ribonucleotide mapping software is customized for data generated using only one type of ribonucleotide sequencing technique. To standardize the analysis of ribonucleotide sequencing experiments, we developed the bioinformatics toolkit, Ribose-Map. Through a series of analytical modules, Ribose-Map transforms raw sequencing data into summary datasets and publication-ready visualizations of results, allowing researchers to locate the genomic coordinates of ribonucleotides to single-nucleotide resolution, study the nucleotide sequence context of ribonucleotides, explore their genome-wide distribution, and identify hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. Ribose-Map is available at https://github.com/agombolay/ribose-map.

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases.

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map.

The most common nonstandard nucleotides found in genomic DNA are ribonucleotides. Although ribonucleotides are frequently incorporated into DNA by replicative DNA polymerases, very little is known about the distribution and signatures of ribonucleotides incorporated into DNA. Recent advances in high-throughput ribonucleotide sequencing can capture the exact locations of ribonucleotides in genomic DNA. Ribose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 h of computing time for most datasets and ~30 min of hands-on time. Ribose-Map is available at https://github.com/agombolay/ribose-map .

Evaluation of repair activity by quantification of ribonucleotides in the genome

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ‐seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ‐seq analysis of cells harboring wild‐type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3′‐shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild‐type and repair‐deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ‐seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ‐seq can provide an estimate of indel risk.

Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases.

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2′ modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

Ribonucleotide incorporation in yeast genomic DNA shows preference for cytosine and guanosine preceded by deoxyadenosine

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

ABSTRACTNucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1–100 carbon ions per point (about 0.3–30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.