Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast.

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to systematically delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We found that deletion of the nucleotide excision repair (NER) genes, RAD1 or RAD14, led to an increase in mutagenesis upon acetaldehyde exposure. Acetaldehyde-induced mutations were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair in Δrad1 strains. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C→A changes coupled with an enrichment of gCn→A changes in the acetaldehyde-treated Δrad1 isolates. The gCn→A mutation signature has been shown to be diagnostic of acetaldehyde exposure in yeast and in human cancers. We also demonstrated that the deletion of the two DNA-protein crosslink (DPC) repair proteases, WSS1 and DDI1, also led to increased acetaldehyde-induced mutagenesis. Defects in base excision repair (BER) led to a mild increase in mutagenesis, while defects in mismatch repair (MMR), homologous recombination repair (HR) and post replicative repair pathways did not impact mutagenesis upon acetaldehyde exposure. Our results in yeast were further corroborated upon analysis of whole exome sequenced liver cancers, wherein, tumors with defects in ERCC1 and ERCC4 (NER), FANCD2 (ICL repair) or SPRTN (DPC repair) carried a higher gCn→A mutation load than tumors with no deleterious mutations in these genes. Our findings demonstrate that multiple DNA repair pathways protect against acetaldehyde-induced mutagenesis.

RNA viruses, M satellites, chromosomal killer genes, and killer/nonkiller phenotypes in the 100-genomes S. cerevisiae strains.

We characterized previously identified RNA viruses (L-A, L-BC, 20S, and 23S), L-A-dependent M satellites (M1, M2, M28, and Mlus), and M satellite-dependent killer phenotypes in the Saccharomyces cerevisiae 100-genomes genetic resource population. L-BC was present in all strains, albeit in 2 distinct levels, L-BChi and L-BClo; the L-BC level is associated with the L-BC genotype. L-BChi, L-A, 20S, 23S, M1, M2, and Mlus (M28 was absent) were in fewer strains than the similarly inherited 2µ plasmid. Novel L-A-dependent phenotypes were identified. Ten M+ strains exhibited M satellite-dependent killing (K+) of at least 1 of the naturally M0 and cured M0 derivatives of the 100-genomes strains; in these M0 strains, sensitivities to K1+, K2+, and K28+ strains varied. Finally, to complement our M satellite-encoded killer toxin analysis, we assembled the chromosomal KHS1 and KHR1 killer genes and used naturally M0 and cured M0 derivatives of the 100-genomes strains to assess and characterize the chromosomal killer phenotypes.

Aldehyde-Associated Mutagenesis─Current State of Knowledge.

Aldehydes are widespread in the environment, with multiple sources such as food and beverages, industrial effluents, cigarette smoke, and additives. The toxic effects of exposure to several aldehydes have been observed in numerous studies. At the molecular level, aldehydes damage DNA, cross-link DNA and proteins, lead to lipid peroxidation, and are associated with increased disease risk including cancer. People genetically predisposed to aldehyde sensitivity exhibit severe health outcomes. In various diseases such as Fanconi's anemia and Cockayne syndrome, loss of aldehyde-metabolizing pathways in conjunction with defects in DNA repair leads to widespread DNA damage. Importantly, aldehyde-associated mutagenicity is being explored in a growing number of studies, which could offer key insights into how they potentially contribute to tumorigenesis. Here, we review the genotoxic effects of various aldehydes, focusing particularly on the DNA adducts underlying the mutagenicity of environmentally derived aldehydes. We summarize the chemical structures of the aldehydes and their predominant DNA adducts, discuss various methodologies, in vitro and in vivo, commonly used in measuring aldehyde-associated mutagenesis, and highlight some recent studies looking at aldehyde-associated mutation signatures and spectra. We conclude the Review with a discussion on the challenges and future perspectives of investigating aldehyde-associated mutagenesis.

A novel narnavirus is widespread in Saccharomyces cerevisiae and impacts multiple host phenotypes.

RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions, and are not known to affect host fitness. In this study, we describe a new S. cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in 1 strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Furthermore, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.

Acetaldehyde makes a distinct mutation signature in single-stranded DNA.

Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG→TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the mutagenic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA treatment is highly mutagenic to cells and leads to strand-biased mutations on guanines (G→T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Polζ. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption. Our study proposes a key mechanism underlying carcinogenesis by acetaldehyde-mutagenesis of single-stranded DNA.

Mitochondrial Genome Variation Affects Multiple Respiration and Nonrespiration Phenotypes in Saccharomyces cerevisiae.

Mitochondrial genome variation and its effects on phenotypes have been widely analyzed in higher eukaryotes but less so in the model eukaryote Saccharomyces cerevisiae Here, we describe mitochondrial genome variation in 96 diverse S. cerevisiae strains and assess associations between mitochondrial genotype and phenotypes as well as nuclear-mitochondrial epistasis. We associate sensitivity to the ATP synthase inhibitor oligomycin with SNPs in the mitochondrially encoded ATP6 gene. We describe the use of iso-nuclear F1 pairs, the mitochondrial genome equivalent of reciprocal hemizygosity analysis, to identify and analyze mitochondrial genotype-dependent phenotypes. Using iso-nuclear F1 pairs, we analyze the oligomycin phenotype-ATP6 association and find extensive nuclear-mitochondrial epistasis. Similarly, in iso-nuclear F1 pairs, we identify many additional mitochondrial genotype-dependent respiration phenotypes, for which there was no association in the 96 strains, and again find extensive nuclear-mitochondrial epistasis that likely contributes to the lack of association in the 96 strains. Finally, in iso-nuclear F1 pairs, we identify novel mitochondrial genotype-dependent nonrespiration phenotypes: resistance to cycloheximide, ketoconazole, and copper. We discuss potential mechanisms and the implications of mitochondrial genotype and of nuclear-mitochondrial epistasis effects on respiratory and nonrespiratory quantitative traits.

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M.

The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed.

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.

The eukaryotic Mcm2-7 replicative helicase.

In eukaryotes, the Mcm2-7 complex forms the core of the replicative helicase - the molecular motor that uses ATP binding and hydrolysis to fuel the unwinding of double-stranded DNA at the replication fork. Although it is a toroidal hexameric helicase superficially resembling better-studied homohexameric helicases from prokaryotes and viruses, Mcm2-7 is the only known helicase formed from six unique and essential subunits. Recent biochemical and structural analyses of both Mcm2-7 and a higher-order complex containing additional activator proteins (the CMG complex) shed light on the reason behind this unique subunit assembly: whereas only a limited number of specific ATPase active sites are needed for DNA unwinding, one particular ATPase active site has evolved to form a reversible discontinuity (gate) in the toroidal complex. The activation of Mcm2-7 helicase during S-phase requires physical association of the accessory proteins Cdc45 and GINS; structural data suggest that these accessory factors activate DNA unwinding through closure of the Mcm2-7 gate. Moreover, studies capitalizing on advances in the biochemical reconstitution of eukaryotic DNA replication demonstrate that Mcm2-7 loads onto origins during initiation as a double hexamer, yet does not act as a double-stranded DNA pump during elongation.