Chemical synthesis of oligonucleotides containing damaged bases for biological studies

Since nucleic acids are organic molecules, even DNA, which carries genetic information, is subjected to various chemical reactions in cells. Alterations of the chemical structure of DNA, which are referred to as DNA damage or DNA lesions, induce mutations in the DNA sequences, which lead to carcinogenesis and cell death, unless they are restored by the repair systems in each organism. Formerly, DNA from bacteria and bacteriophages and DNA fragments treated with UV or gamma radiation, alkylating or crosslinking agents, and other carcinogens were used as damaged DNA for biochemical studies. With these materials, however, it is difficult to understand the detailed mechanisms of mutagenesis and DNA repair. Recent progress in the chemical synthesis of oligonucleotides has enabled us to incorporate a specific lesion at a defined position within any sequence context. This method is especially important for studies on mutagenesis and translesion synthesis, which require highly pure templates, and for the structural biology of repair enzymes, which necessitates large amounts of substrate DNA as well as modified substrate analogs. In this review, the various phosphoramidite building blocks for the synthesis of lesion-containing oligodeoxyribonucleotides are described, and some examples of their applications to molecular and structural biology are presented.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis

Abstract

Environmental mutagens lead to mutagenesis. However, the mechanisms are very complicated and not fully understood. Environmental mutagens produce various DNA lesions, including base-damaged or sugar-modified DNA lesions, as well as epigenetically modified DNA. DNA polymerases produce mutation spectra in translesion DNA synthesis (TLS) through misincorporation of incorrect nucleotides, frameshift deletions, blockage of DNA replication, imbalance of leading- and lagging-strand DNA synthesis, and genome instability. Motif or subunit in DNA polymerases further affects the mutations in TLS. Moreover, protein interactions and accessory proteins in DNA replisome also alter mutations in TLS, demonstrated by several representative DNA replisomes. Finally, in cells, multiple DNA polymerases or cellular proteins collaborate in TLS and reduce in vivo mutagenesis. Summaries and perspectives were listed. This review shows mechanisms of mutagenesis induced by DNA lesions and the effects of multiple factors on mutations in TLS in vitro and in vivo.     

Replication of Damaged DNA and the Molecular Mechanism of Ultraviolet Light Mutagenesis

Abstract

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.     

Base Excision Repair and its Role in Maintaining Genome Stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10⁴ events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Combining structural and bioinformatics methods for the analysis of functionally important residues in DNA glycosylases

An essential function of DNA glycosylases is the recognition and excision of damaged bases in DNA, thereby preserving genomic integrity. Lesion recognition is a multistep process, which is only partially revealed by structural analysis of the catalytically competent complex. The functional role of additional residues can be predicted by combining structural data with analysis of amino acid conservation. The following postulate underlies this approach: if a family or superfamily can be broken into subgroups with different substrate specificities, residues highly conserved between these subgroups represent those important for enzyme catalysis and structure maintenance while residues highly conserved within a subgroup but not between subgroups represent residues important for substrate specificity. We review the bioinformatics approach used for this quantitative analysis and describe its application to the Nth superfamily and Fpg family of DNA glycosylases. These results serve as a starting point in planning site-directed mutagenesis experiments to elucidate the functional role of similar and dissimilar residues in DNA repair and other proteins.     

Synthesis of oligodeoxynucleotides containing a single diastereoisomer of alpha-(N2-2'-deoxyguanosinyl)-N-desmethyltamoxifen

A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was described. The site-specifically modified oligodeoxynucleotide can be used for mutagenesis, DNA repair, and 3D structural studies and also as standard for quantitative analysis of TAM-DNA adducts in animal and human.     

Uracil‐DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes

Uracil‐DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil‐DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.     

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases have been found in various eukaryotes, ranging from yeasts to mammals, including humans. According to partial homology of the primary structure, they are grouped into families B, X, and Y. These enzymes display a high infidelity on an intact DNA template, but they are accurate on a damaged template. Error-prone DNA polymerases are characterized by probabilities of base substitution or frameshift mutations ranging from 10^?3 to 7.5 · 10^?1 in an intact DNA, whereas the spontaneous mutagenesis rate per replicated nucleotide varies between 10^?10 and 10^?12. Low-fidelity polymerases are terminal deoxynucleotidyl transferase (TdT) and DNA polymerases β, ζ, κ, η, ι, λ, μ, and Rev1. The main characteristics of these enzymes are reviewed. None of them exhibits proofreading 3′ → 5′ exonuclease (PE) activity. The specialization of these polymerases consists in their capacity for synthesizing opposite DNA lesions (not eliminated by the numerous repair systems), which is explained by the flexibility of their active centers or a limited ability to express TdT activity. Classic DNA polymerases α, δ, ε, and γ cannot elongate primers with mismatched nucleotides at the 3′-end (which leads to replication block), whereas some specialized polymerases can catalyze this elongation. This is accompanied by overcoming the replication block, often at the expense of an increased mutagenesis rate. How can a cell exist under the conditions of this high infidelity of many DNA polymerase activities? Not all tissues of the body contain a complete set of low-fidelity DNA polymerases, although some of these enzymes are vitally important. In addition, cells “should not allow” error-prone DNA polymerases to work on undamaged DNA. After a lesion on the DNA template is bypassed, the cell should switch over from DNA synthesis catalyzed by specialized polymerases to the synthesis catalyzed by relatively high-fidelity DNA polymerases δ and ? (with an error frequency of 10^?5 to 10^?6) as soon as possible. This is done by forming complexes of polymerase δ or ? with proliferating cell nuclear antigen (PCNA) and replication factors RP-A and RF-C. These highly processive complexes show a greater affinity to correct primers than specialized DNA polymerases do. The fact that specialized DNA polymerases are distributive or weakly processive favors the switching. The fidelity of these polymerases is increased by the PE function of DNA polymerases δ and ε, as well as autonomous 3′ → 5′ exonucleases, which are widespread over the entire phylogenetic tree of eukaryotes. The exonuclease correction decelerates replication in the presence of lesions in the DNA template but increases its fidelity, which decreases the probability of mutagenesis and carcinogenesis.     

Measurement of oxidatively induced DNA damage and its repair,by mass spectrometric techniques

Abstract

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5’-cyclopurine-2’-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.     

Having a direct look: Analysis of DNA damage and repair mechanisms by next generation sequencing

Genetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis.     

A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.     

Translesion DNA polymerases in eukaryotes: what makes them tick?

Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. “Hassle-free” DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges. Among the most pressing obstacles that replicative polymerases often cannot overcome by themselves are lesions that distort the structure of DNA. Despite elaborate systems that cells utilize to cleanse their genomes of damaged DNA, repair is often incomplete. The persistence of DNA lesions obstructing the cellular replicases can have deleterious consequences. One of the mechanisms allowing cells to complete replication is “Translesion DNA Synthesis (TLS)”. TLS is intrinsically error-prone, but apparently, the potential downside of increased mutagenesis is a healthier outcome for the cell than incomplete replication. Although most of the currently identified eukaryotic DNA polymerases have been implicated in TLS, the best characterized are those belonging to the “Y-family” of DNA polymerases (pols η, ι, κ and Rev1), which are thought to play major roles in the TLS of persisting DNA lesions in coordination with the B-family polymerase, pol ζ. In this review, we summarize the unique features of these DNA polymerases by mainly focusing on their biochemical and structural characteristics, as well as potential protein–protein interactions with other critical factors affecting TLS regulation.     

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences.     

Dependence of the viability and mutability of Escherichia coli K-12 cells on the balance of DNA and protein syntheses. II. The effect of disturbance in the balance of DNA and protein syntheses on the cellular mutability and viability of rec+ and rec- strains treated with nalidixic acid]

The changes in the survival and mutagenesis of rec+ and rec- Escherichia coli K-12 strains, treated with the selective inhibitor of DNA synthesis, nalidixic acid, are found to be due to the processes of the stabilization and repair of the metabolic gaps in DNA chains, which depend on the balance of DNA and protein synthesis. The various character is observed of the relation between the survival and the mutagenesis and the balance of DNA and protein syntheses in cells which are valuable (rec+) and defective (rec-) for the processes of DNA repair.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

Activities of chlorinated ethane and ethylene compounds in the Salmonella/rat microsome mutagenesis and rat hepatocyte/DNA repair assays under vapor phase exposure conditions

Three chlorinated ethane and ethylene solvent products were examined for their genotoxicity in the Salmonella/microsome mutagenesis and hepatocyte primary culture DNA repair assays using vapor phase exposures. The positive control in this study, monochloroethylene (vinyl chloride), induced reversion mutation of Salmonella tester strains TA100 and TA1535 with enhancement by an exogenous activation system and elicited unscheduled DNA synthesis in rat hepatocytes in culture. Exposures to 1,1,1-trichloroethane (methyl chloroform) or 1,1,2-trichloroethylene samples which contained stabilizers resulted in increased recovery of revertant colonies of Salmonella at concentrations causing greater than 96% cell killing. However, these stabilized materials did not induce DNA repair and low-stabilized trichloroethylene did not induce reversion mutation or DNA repair. Exposure of Salmonella tester strains and hepatocytes to highly toxic vapor concentrations of technical grade 1,1,2,2-tetrochloroethylene, low-stabilized and stabilized, increased reversion mutation and elicited DNA repair. Tetrachloroethylene of high purity was not genotoxic. With all of these test products, the presence of an Aroclor-induced rat liver subcellular enzyme preparation in the mutagenesis assay did not have any effect on the results. These observations suggest that stabilizers or unknown impurities normally present at low concentrations in these products are responsible for the positive responses observed at the high exposure concentrations achievable under in vitro test conditions.Abbreviation HPC hepatocyte primary culture