A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in <i>Caenorhabditis elegans</i>

Ionizing radiation (IR) is commonly used in cancer therapy and is a main source of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA damage. We have used Caenorhabditis elegans as an invertebrate model to identify novel factors required for repair of DNA damage inflicted by IR. We have performed an unbiased genetic screen, finding that smg-1 mutations confer strong hyper-sensitivity to IR. SMG-1 is a phosphoinositide-3 kinase (PI3K) involved in mediating nonsense-mediated mRNA decay (NMD) of transcripts containing premature stop codons and related to the ATM and ATR kinases which are at the apex of DNA damage signaling pathways. Hyper-sensitivity to IR also occurs when other genes mediating NMD are mutated. The hyper-sensitivity to bleomycin, a drug known to induce DSBs, further supports that NMD pathway mutants are defective in DSB repair. Hyper-sensitivity was not observed upon treatment with alkylating agents or UV irradiation. We show that SMG-1 mainly acts in mitotically dividing germ cells, and during late embryonic and larval development. Based on epistasis experiments, SMG-1 does not appear to act in any of the three major pathways known to mend DNA DSBs, namely homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ). We speculate that SMG-1 kinase activity could be activated following DNA damage to phosphorylate specific DNA repair proteins and/or that NMD inactivation may lead to aberrant mRNAs leading to synthesis of malfunctioning DNA repair proteins

C. elegans genome-wide analysis reveals DNA repair pathways that act cooperatively to preserve genome integrity upon ionizing radiation.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions

Systemic influences of mammary cancer on monocytes in mice

SIMPLE SUMMARY: Using a mouse model of breast cancer driven by the mammary epithelial expression of the polyoma middle T oncoprotein in which the tumors progress from benign to malignant metastatic stages, we show that cancer causes an increase in circulating monocytes and a splenomegaly. This increase in monocyte number is due to their increased proliferation in the bone marrow and not turnover rates in the blood. Single cell sequencing also shows that new populations of monocytes do not arise during cancer. Cancer also drives systemic changes in the monocyte transcriptome, with a notable down-regulation of interferon signaling. These systemic influences start in the bone marrow but intensify in the blood. Comparison of cancer prone and cancer resistant mouse inbred strains carrying the same oncogene reveals that the genetic background of the strain causes different monocyte transcriptional changes. Similarly, a comparison of the mouse transcriptome to human breast cancer monocyte profiles indicates limited similarities, to the extent that interferon signaling is enhanced in humans. Systemic responses are different in the same model of cancer on different genetic backgrounds within a species and even greater changes are found across species. These data suggest that at the very least this mouse model will be limited when it comes to exploring the mechanism behind systemic changes in humans. ABSTRACT: There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer

Protection of the C. elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis

Mutational signatures are jointly shaped by DNA damage and repair.

Funder: Biotechnology and Biological Sciences Research Council East of Scotland Bioscience Doctoral Training Partnership Ph.D. studentshipFunder: Korean Institute for Basic Science, IBS-R022-A1-2019Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes

The push–pull mechanism of bacteriophage Ø29 DNA injection

The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well-established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right-left polarity, in a two-step process. In the first step ∼65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy-dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.This work was supported by research grants 2R01 G27242-24 from the National Institutes of Health, BMC2002-03818 from Dirección General de Investigación and by an Institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’.Peer reviewe

Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control

Protein p6 of Bacillus subtilis bacteriophage phi 29 is essential for phage development. In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to phi 29 DNA in vivo. By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology. In conclusion, binding of protein p6 all along the phi 29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein. The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of phi 29 DNAThis work was supported by research grants 2R01 GM27242-24 from the National Institutes of Health, BMC2002-03818 from Ministerio de Ciencia y Tecnología and by an Institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.G.-H. is the recipient of a postdoctoral fellowship of Comunidad Autónoma de MadridPeer reviewe

Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control

Protein p6 of Bacillus subtilis bacteriophage phi 29 is essential for phage development. In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to phi 29 DNA in vivo. By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology. In conclusion, binding of protein p6 all along the phi 29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein. The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of phi 29 DNAThis work was supported by research grants 2R01 GM27242-24 from the National Institutes of Health, BMC2002-03818 from Ministerio de Ciencia y Tecnología and by an Institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.G.-H. is the recipient of a postdoctoral fellowship of Comunidad Autónoma de MadridPeer reviewe

Requeriment for Bacillus subtilis bacteriophage ø29 DNA ejection

Phage Φ29 infects Bacillus subtilis and ejects its linear DNA with a right to left polarity in a two-step, “push–pull” mechanism. In the first step 65% of the DNA is pushed inside the cell, presumably by the pressure built inside the capsid. In the second step, the remaining DNA is pulled by a hypothetical motor that comprises at least viral protein p17, encoded by the right early operon, in an energy-dependent process. We have further studied Φ29 DNA ejection by using energy poisons and DNA replication and transcription inhibitors. The first step is passive, as it does not require an external energy source. The second step is transcription-independent and is completely abolished by novobiocin, suggesting a requirement for negatively supercoiled DNA. Viral DNA pulling also requires an electrochemical proton gradient, as the process is highly impaired by specific energy poisons such as gramicidin and CCCP (carbonyl cyanide m-chlorophenylhydrazone). The fact that azide has no effect in the absence of p17 suggests that this protein is essential for energy transduction.This work was supported by research grants BMC2002-03818 from Dirección General de Investigación (to M.S.) and GR/SAL/0179/2004 from Comunidad Autónoma de Madrid (to J.M.H.) and by an Institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”. V.G.-H. was a recipient of a postdoctoral fellowship of the Comunidad Autónoma de Madrid.Peer reviewe