Search

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin (NCL) is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that the yeast nucleolin Nsr1 is enriched at a G4 DNA-forming sequence in vivo and is a major factor in inducing the genome instability associated with the cotranscriptionally formed G4 DNA in the yeast genome. We also show that Nsr1 results in impeding replication past such a G4 DNA-forming sequence. The G4-associated genome instability and the G4 DNA-binding in vivo require the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of the RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vivo and restores unhindered replication, overall resulting in a sharp reduction in the genome instability associated with a guanine-rich G4 DNA-forming sequence. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and impeding replication.

G4 DNA is a non-canonical DNA structure consisting of a stacked array of Gquartets held together by base pairing between guanine bases. The formation of G4 DNA requires a cluster of guanine-runs within a strand of DNA. Even though the chemistry of this remarkable DNA structure has been under investigation for decades, evidence supporting the biological relevance of G4 DNA has only begun to emerge and point to very important and conserved biological functions. This review will specifically focus on the interplay between transcription and G4 DNA and discuss two alternative but interconnected perspectives. The first part of the review will describe the evidence substantiating the intriguing idea that a shift in DNA structural conformation could be another layer of non-genetic or epigenetic regulator of gene expression and thereby an important determinant of cell fate. The second part will describe the recent genetic studies showing that those genomic loci containing G4 DNA-forming guanine-rich sequences are potential hotspots of genome instability and that the level and orientation of transcription is critical in the materialization of genome instability associated with these sequences.

Non-canonical residue in DNA is a major and conserved source of genome instability. The appearance of uracil residues in DNA accompanies a significant mutagenic consequence and is regulated at multiple levels, from the concentration of available dUTP in the nucleotide pool to the excision repair for removal from DNA. Recently, an interesting phenomenon of transcription-associated elevation in uracil-derived mutations was described in Saccharomyces cerevisiae genome. While trying to understand the variability in mutagenesis, we uncovered that the frequency of uracil incorporation into DNA can vary depending on the transcription rate and that the non-replicative, repair-associated DNA synthesis underlies the higher uracil density of the actively transcribed genomic loci. This novel mechanism brings together the chemical vulnerability of DNA under transcription and the uracil-associated mutagenesis, and has the potential to apply to other non-canonical residues of mutagenic importance. Keyword: DNA repair; Non-canonical nucleotides; Transcription-associated mutagenesis; Uracil dUTPase

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

2019 Klein et al., Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies., HLK was supported by NIH grant R01-CA146940. AA was supported by the European research Council (ERC2014- ADG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), and the Junta de Andalucía (BIO1238). JLA was supported by NIH grant R35G-GM119788. CHF was supported by NIH grants P01- GM105473 and R01-GM122880. SMG was supported by the Swiss national Science Foundation and the Novartis Research Foundation. DAG was supported by National Institutes of Health (Intramural Research Program Project Z1AES103266. JEH was supported by NIH grants R35- GM127029 and P01-GM105473. GI was supported by NIH grants R01-GM125650 and R01-GM080600. SJ-R was supported by NIH grant R35-GM118077. MCK was supported by National Science Foundation Graduate Research Fellowship DGE-1122492, NIH training grant T32-GM007223 and NIH grant DP20D008429. RDK and CDP were supported by NIH grants R01-GM26017 and R01-GM50006. AK was supported by NIH grant R01-GM073115. SAEL was supported by grants from Agence Nationale de la Recherche ANR-14- CE10-0010-01 and the Foundation pour la Recherche Médicale Equipe FRM DEQ20160334889. SEL was supported by NIH grant R01-GM071011. SMM was supported by NIH grants R01-GM60987 and P01-GM105473. TDP was supported by NIH grant R35-GM118020. SMR was supported by a gift from WM Keck Foundation, NIH Director’s Pioneer Award DP1-CA174424 and NIH grant R35-GM122598. DMF was supported by the Cancer Prevention and Research Institute of Texas, Baylor College of Medicine Comprehensive Cancer Training Program Postdoctoral Fellowship RP160283. RR was supported by NIH grant R35-GM118180 and JM-H was supported by a Marie Curie International Outgoing Fellowship and the ANR-12-PDOC-0035-01. LSS was supported by NIH grant R35-GM126997. PZ was supported by National Science Centre Poland 2015/19/103859 and FNP First TEAM/2016-1/9. NK was supported by NIH grant R01-GM116007 and the Welch Foundation (AU1875). ML was supported by grants from the Danish Agency for Science, Technology and Innovation, the Villum Foundation and the Danish National Research Foundation (DNRF115). AM was supported by NIH grants R35-GM127006 and R03- ES029306.

Recombination and mutagenesis are elevated by active transcription. The correlation between transcription and genome instability is largely explained by the topological and structural changes in DNA and the associated physical obstacles generated by the transcription machinery. However, such explanation does not directly account for the unique types of mutations originating from the non-canonical residues, uracil or ribonucleotide, which are also elevated at highly transcribed regions. Based on the previous findings that abasic (AP) lesions derived from the uracil residues incorporated into DNA in place of thymine constitute a major component of the transcription-associated mutations in yeast, we formed the hypothesis that DNA synthesis ensuing from the repair of the transcription-induced DNA damage provide the opportunity for uracil-incorporation. In support of this hypothesis, we show here the positive correlation between the level of transcription and the density of uracil residues in the yeast genome indirectly through the mutations generated by the glycosylase that excise undamaged cytosine as well as uracil. The higher uracil-density at actively transcribed regions is confirmed by the long-amplicon PCR analysis. We also show that the uracil-associated mutations at a highly transcribed region are elevated by the induced DNA damage and reduced by the overexpression of a dUTP-catalyzing enzyme Dut1 in G1- or G2-phases of the cell cycle. Overall, our results show that the DNA composition can be modified to include higher uracil-content through the non-replicative, repair-associated DNA synthesis., Author summary Uracil in DNA, a major source of spontaneous mutations, can occur through the deamination of cytosine residues or through the direct incorporation of dUTP by DNA polymerases. Recent studies in yeast have shown that the uracil-associated mutations occur more frequently at highly transcribed regions. Because the reduction in dUTP pool decreased these mutations, it was postulated that the extent of uracil-incorporation into DNA is significantly affected by the local transcription activity. We show here that the higher transcription rate does correlate with the higher uracil-density in the yeast genome. We further provide multiple lines of evidence supporting a model of uracil-incorporation into DNA that is dependent on the repair synthesis of transcription-associated DNA damage.

Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation.

G-quadruplexes (G4) are higher-order DNA and RNA secondary structures formed by G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential G4 quadruplex sequences have been identified in G-rich eukaryotic non-telomeric and telomeric genomic regions. Upon function, G4 formation is known to involve in chromatin remodelling, gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. The natural role and biological validation of G4 structures is starting to be explored, and is of particular interest for the therapeutic interventions for human diseases. However, the existence and physiological role of G4 DNA and G4 RNA in plants species have not been much investigated yet and therefore, is of great interest for the development of improved crop varieties for sustainable agriculture. In this context, several recent studies suggests that these highly diverse G4 structures in plants can be employed to regulate expression of genes involved in several pathophysiological conditions including stress response to biotic and abiotic stresses as well as DNA damage. In the current review, we summarize the recent findings regarding the emerging functional significance of G4 structures in plants and discuss their potential value in the development of improved crop varieties.

Topoisomerases manage the torsional stress associated with the separation of DNA strands during transcription and DNA replication. Eukaryotic Topoisomerase I (Top1) is a Type IB enzyme that nicks and rejoins only one strand of duplex DNA, and it is especially important during transcription. By resolving transcription-associated torsional stress, Top1 reduces the accumulation of genome-destabilizing R-loops and non-B DNA structures. The DNA nicking activity of Top1, however, can also initiate genome instability in the form of illegitimate recombination, homologous recombination and mutagenesis. In this review, we focus on the diverse, and often opposing, roles of Top1 in regulating eukaryotic genome stability.

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA.

Alterations in genome sequence and structure contribute to somatic disease, affect the fitness of subsequent generations and drive evolutionary processes. The crucial roles of highly accurate replication and efficient repair in maintaining overall genome integrity are well-known, but the more localized stability costs that are associated with transcribing DNA into RNA molecules are less appreciated. Here we review the diverse ways in which the essential process of transcription alters the underlying DNA template and thereby modifies the genetic landscape.

Non-B DNA structures are a major contributor to the genomic instability associated with repetitive sequences. Immunoglobulin switch Mu (Sμ) region sequence is comprised of guanine-rich repeats and has high potential for forming G4 DNA, in which one strand of DNA folds into an array of guanine quartets. Taking advantage of the genetic tractability of Saccharomyces cerevisiae, we developed a recombination assay to investigate mechanisms involved in maintaining stability of G-rich repetitive sequence. By embedding Sμ sequence within recombination substrates under the control of a tetracycline-regulatable promoter, we demonstrate that the rate and orientation of transcription both affect the stability of Sμ sequence. In particular, the greatest instability was observed under high-transcription conditions when the Sμ sequence was oriented with the C-rich strand as the transcription template. The effect of transcription orientation was enhanced in the absence of the Type IB topoiosmerase Top1, possibly due to enhanced R-loop formation. Loss of Sgs1 helicase and RNase H activity also increased instability, suggesting they may cooperatively function to reduce the formation of non-B DNA structures in highly transcribed regions. Finally, the Sμ sequence was unstable when transcription elongation was perturbed due to a defective THO complex. In a THO-deficient background, there was further exacerbation of orientation-dependent instability associated with the ectopically expressed, single-strand cytosine deaminase AID. The implications of our findings to understanding instability associated with potential G4 DNA forming sequences are discussed.

A high level of transcription has been associated with elevated spontaneous mutation and recombination rates in eukaryotic organisms. To determine whether the transcription level is directly correlated with the degree of genomic instability, we have developed a tetracycline-regulated LYS2 reporter system to modulate the transcription level over a broad range in Saccharomyces cerevisiae. We find that spontaneous mutation rate is directly proportional to the transcription level, suggesting that movement of RNA polymerase through the target initiates a mutagenic process(es). Using this system, we also investigated two hypotheses that have been proposed to explain transcription-associated mutagenesis (TAM): (1) transcription impairs replication fork progression in a directional manner and (2) DNA lesions accumulate under high-transcription conditions. The effect of replication fork progression was probed by comparing the mutational rates and spectra in yeast strains with the reporter gene placed in two different orientations near a well-characterized replication origin. The effect of endogenous DNA damage accumulation was investigated by studying TAM in strains defective in nucleotide excision repair or in lesion bypass by the translesion polymerase Polzeta. Our results suggest that both replication orientation and endogenous lesion accumulation play significant roles in TAM, particularly in terms of mutation spectra.

The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination.

Somatic hypermutation of Ig genes is probably dependent on transcription of the target gene via a mutator factor associated with the RNA polymerase (Storb, U., E.L. Klotz, J. Hackett, Jr., K. Kage, G. Bozek, and T.E. Martin. 1998. J. Exp. Med. 188:689–698). It is also probable that some form of DNA repair is involved in the mutation process. It was shown that the nucleotide excision repair proteins were not required, nor were mismatch repair (MMR) proteins. However, certain changes in mutation patterns and frequency of point mutations were observed in Msh2 (MutS homologue) and Pms2 (MutL homologue) MMR-deficient mice (for review see Kim, N., and U. Storb. 1998. J. Exp. Med. 187:1729–1733). These data were obtained from endogenous immunoglobulin (Ig) genes and were presumably influenced by selection of B cells whose Ig genes had undergone certain mutations. In this study, we have analyzed somatic hypermutation in two MutL types of MMR deficiencies, Pms2 and Mlh1. The mutation target was a nonselectable Ig-κ gene with an artificial insert in the V region. We found that both Pms2- and Mlh1-deficient mice can somatically hypermutate the Ig test gene at approximately twofold reduced frequencies. Furthermore, highly mutated sequences are almost absent. Together with the finding of genome instability in the germinal center B cells, these observations support the conclusion, previously reached for Msh2 mice, that MMR-deficient B cells undergoing somatic hypermutation have a short life span. Pms2- and Mlh1-deficient mice also resemble Msh2-deficient mice with respect to preferential targeting of G and C nucleotides. Thus, it appears that the different MMR proteins do not have unique functions with respect to somatic hypermutation. Several intrinsic characteristics of somatic hypermutation remain unaltered in the MMR-deficient mice: a preference for targeting A over T, a strand bias, mutational hot spots, and hypermutability of the artificial insert are all seen in the unselectable Ig gene. This implies that the MMR proteins are not required for and most likely are not involved in the primary step of introducing the mutations. Instead, they are recruited to repair certain somatic point mutations, presumably soon after these are created.

G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼20 kb or ∼100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed., Author Summary Genome instability is not evenly distributed, but rather is highly elevated at certain genomic loci containing DNA sequences that can fold into non-canonical secondary structures. The four-stranded G-quadruplex or G4 DNA is one such DNA structure capable of instigating transcription and/or replication obstruction and subsequent genome instability. In this study, we used a reporter system to quantitatively measure the level of genome instability occurring at a G4 DNA motif integrated into the yeast genome. We showed that the disruption of Topoisomerase I function significantly elevated various types of genome instability at the highly transcribed G4 motif generating loss of heterozygosity and copy number alterations (deletions and duplications), both of which are frequently observed in cancer genomes.