Methods to Study Z-DNA-Induced Genetic Instability.

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic and eukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems.

Dynamic alternative DNA structures in biology and disease.

Repetitive elements in the human genome, once considered 'junk DNA', are now known to adopt more than a dozen alternative (that is, non-B) DNA structures, such as self-annealed hairpins, left-handed Z-DNA, three-stranded triplexes (H-DNA) or four-stranded guanine quadruplex structures (G4 DNA). These dynamic conformations can act as functional genomic elements involved in DNA replication and transcription, chromatin organization and genome stability. In addition, recent studies have revealed a role for these alternative structures in triggering error-generating DNA repair processes, thereby actively enabling genome plasticity. As a driving force for genetic variation, non-B DNA structures thus contribute to both disease aetiology and evolution.

Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

Distinct mechanisms of mutagenic processing of alternative DNA structures by repair proteins.

Repetitive sequences can form a variety of alternative DNA structures (non-B DNA) that can modulate transcription, replication, and repair. However, non-B DNA-forming sequences can also stimulate mutagenesis, and are enriched at mutation hotspots in human cancer genomes. Interestingly, different types of non-B DNA stimulate mutagenesis via distinct repair processing mechanisms.

Distinct DNA repair pathways cause genomic instability at alternative DNA structures.

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF-dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the sub-molecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.

Modulation of DNA structure formation using small molecules.

Genome integrity is essential for proper cell function such that genetic instability can result in cellular dysfunction and disease. Mutations in the human genome are not random, and occur more frequently at "hotspot" regions that often co-localize with sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures. Non-B DNA-forming sequences are mutagenic, can stimulate the formation of DNA double-strand breaks, and are highly enriched at mutation hotspots in human cancer genomes. Thus, small molecules that can modulate the conformations of these structure-forming sequences may prove beneficial in the prevention and/or treatment of genetic diseases. Further, the development of molecular probes to interrogate the roles of non-B DNA structures in modulating DNA function, such as genetic instability in cancer etiology are warranted. Here, we discuss reported non-B DNA stabilizers, destabilizers, and probes, recent assays to identify ligands, and the potential biological applications of these DNA structure-modulating molecules.

DNA fragility in the parallel evolution of pelvic reduction in stickleback fish.

Evolution generates a remarkable breadth of living forms, but many traits evolve repeatedly, by mechanisms that are still poorly understood. A classic example of repeated evolution is the loss of pelvic hindfins in stickleback fish (Gasterosteus aculeatus). Repeated pelvic loss maps to recurrent deletions of a pelvic enhancer of the Pitx1 gene. Here, we identify molecular features contributing to these recurrent deletions. Pitx1 enhancer sequences form alternative DNA structures in vitro and increase double-strand breaks and deletions in vivo. Enhancer mutability depends on DNA replication direction and is caused by TG-dinucleotide repeats. Modeling shows that elevated mutation rates can influence evolution under demographic conditions relevant for sticklebacks and humans. DNA fragility may thus help explain why the same loci are often used repeatedly during parallel adaptive evolution.

Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases.

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability.

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease.

Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development.

New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity.

Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA-RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA-RNA and RNA-RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA-RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review.

Short Inverted Repeats Are Hotspots for Genetic Instability: Relevance to Cancer Genomes.

Analyses of chromosomal aberrations in human genetic disorders have revealed that inverted repeat sequences (IRs) often co-localize with endogenous chromosomal instability and breakage hotspots. Approximately 80% of all IRs in the human genome are short (<100 bp), yet the mutagenic potential of such short cruciform-forming sequences has not been characterized. Here, we find that short IRs are enriched at translocation breakpoints in human cancer and stimulate the formation of DNA double-strand breaks (DSBs) and deletions in mammalian and yeast cells. We provide evidence for replication-related mechanisms of IR-induced genetic instability and a novel XPF cleavage-based mechanism independent of DNA replication. These discoveries implicate short IRs as endogenous sources of DNA breakage involved in disease etiology and suggest that these repeats represent a feature of genome plasticity that may contribute to the evolution of the human genome by providing a means for diversity within the population.

Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.

Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability.

Guanine holes are prominent targets for mutation in cancer and inherited disease.

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells.

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

Methods to detect replication-dependent and replication-independent DNA structure-induced genetic instability.

DNA can adopt a variety of alternative secondary (i.e., non-B DNA) conformations that play important roles in cellular metabolism, including genetic instability, disease etiology and evolution. While we still have much to learn, research in this field has expanded dramatically in the past decade. We have summarized in our previous Methods review (Wang et al., Methods, 2009) some commonly used techniques to determine non-B DNA structural conformations and non-B DNA-induced genetic instability in prokaryotes and eukaryotes. Since that time, we and others have further characterized mechanisms involved in DNA structure-induced mutagenesis and have proposed both replication-dependent and replication-independent models. Thus, in this review, we highlight some current methodologies to identify DNA replication-related and replication-independent mutations occurring at non-B DNA regions to allow for a better understanding of the mechanisms underlying DNA structure-induced genetic instability. We also describe a new web-based search engine to identify potential intramolecular triplex (H-DNA) and left-handed Z-DNA-forming motifs in entire genomes or at selected sequences of interest.

The yin and yang of repair mechanisms in DNA structure-induced genetic instability.

DNA can adopt a variety of secondary structures that deviate from the canonical Watson-Crick B-DNA form. More than 10 types of non-canonical or non-B DNA secondary structures have been characterized, and the sequences that have the capacity to adopt such structures are very abundant in the human genome. Non-B DNA structures have been implicated in many important biological processes and can serve as sources of genetic instability, implicating them in disease and evolution. Non-B DNA conformations interact with a wide variety of proteins involved in replication, transcription, DNA repair, and chromatin architectural regulation. In this review, we will focus on the interactions of DNA repair proteins with non-B DNA and their roles in genetic instability, as the proteins and DNA involved in such interactions may represent plausible targets for selective therapeutic intervention.

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells.

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.

Pathways for double-strand break repair in genetically unstable Z-DNA-forming sequences.

DNA can adopt many structures that differ from the canonical B-form, and several of these non-canonical DNA structures have been implicated in genetic instability associated with human disease. Earlier, we found that Z-DNA causes DNA double-strand breaks (DSBs) in mammalian cells that can result in large-scale deletions and rearrangements. In contrast, the same Z-DNA-forming CG repeat in Escherichia coli resulted in only small contractions or expansions within the repeat. This difference in the Z-DNA-induced mutation spectrum between mammals and bacteria might be due to different mechanisms for DSB repair; in mammalian cells, non-homologous end-joining (NHEJ) is a major DSB repair pathway, while E. coli do not contain this system and typically use homologous recombination (HR) to process DSBs. To test the extent to which the different DSB repair pathways influenced the Z-DNA-induced mutagenesis, we engineered bacterial E.coli strains to express an inducible NHEJ system, to mimic the situation in mammalian cells. Mycobacterium tuberculosis NHEJ proteins Ku and ligase D (LigD) were expressed in E.coli cells in the presence or absence of HR, and the Z-DNA-induced mutations were characterized. We found that the presence of the NHEJ mechanism markedly shifted the mutation spectrum from small deletions/insertions to large-scale deletions (from 2% to 24%). Our results demonstrate that NHEJ plays a role in the generation of Z-DNA-induced large-scale deletions, suggesting that this pathway is associated with DNA structure-induced destabilization of genomes from prokaryotes to eukaryotes.