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 γ 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.     

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.     

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 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.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

Dual role of NER in mutagenesis in Pseudomonas putida

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida-besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.     

Multiple DNA damage recognition factors involved in mammalian nucleotide excision repair

The nucleotide excision repair (NER) subpathway operating throughout the mammalian genome is a versatile DNA repair system that can remove a wide variety of helix-distorting base lesions. This system contributes to prevention of blockage of DNA replication by the lesions, thereby suppressing mutagenesis and carcinogenesis. Therefore, it is of fundamental significance to understand how the huge genome can be surveyed for occurrence of a small number of lesions. Recent studies have revealed that this difficult task seems to be accomplished through sequential actions of multiple DNA damage recognition factors, including UV-DDB, XPC, and TFIIH. Notably, these factors adopt completely different strategies to recognize DNA damage. XPC detects disruption and/or destabilization of the base pairing, which ensures a broad spectrum of substrate specificity for global genome NER. In contrast, UV-DDB directly recognizes particular types of lesions, such as UV-induced photoproducts, thereby vitally recruiting XPC as well as further extending the substrate specificity. After DNA binding by XPC, moreover, the helicase activity associated with TFIIH scans a DNA strand to make a final search for the presence of aberrant chemical modifications of DNA. The combination of these different strategies makes a crucial contribution to simultaneously achieving efficiency, accuracy, and versatility of the entire repair system.     

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.     

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.     

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.     

Uracil DNA glycosylase: insights from a master catalyst.

The recognition and removal of damaged bases in the genome is the province of a highly specialized assemblage of enzymes known as DNA glycosylases. In recent years, structural and mechanistic studies have rapidly moved forward such that in some cases, the high-resolution structures of all stable complexes along the reaction pathway are available. In parallel, advances in isotopic labeling of DNA have allowed the determination of a transition state structure of a DNA repair glycosylase using kinetic isotope effect methods. The use of stable substrate analogs and fluorescent probes have provided methods for real time measurement of the critical step of damaged base flipping. Taken together, these synergistic structural and chemical approaches have elevated our understanding of DNA repair enzymology to the level previously attained in only a select few enzymatic systems. This review summarizes recent studies of the paradigm enzyme, uracil DNA glycosylase, and discusses future areas for investigation in this field.     

Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.     

Mechanisms of clastogen-induced chromosomal aberrations: A critical review and description of a model based on failures of tethering of DNA strand ends to strand-breaking enzymes

Most theories of the mechanisms of chromosomal aberrations involve the concepts of clastogens directly acting on DNA to produce strand breaks, and subsequently, the survival of these directly caused DNA strand breaks – or misrepairs of them – through to metaphase when they appear as chromosomal ‘breaks’ or translocations. Nevertheless, various observations are inconsistent with these theories such as the fact that many chemical clastogens (e.g. caffeine, acridines) do not covalently react with DNA, while almost all of the chemical clastogens (e.g. alkylating agents) which do react covalently with DNA, do not directly cause DNA strand breaks. This paper reviews the ‘direct-clastogen damage to DNA’ theories, and the phenomenology of chromosomal aberrations which are inconsistent with them. Then the theory is considered that the breaks in chromosomes seen at metaphase and anaphase are not the survivors of DNA breaks directly induced by clastogens, but rather derive from breaks created by the enzymes which repair damaged DNA. After that, newer knowledge is reviewed that (i) strand breaks are created during normal DNA unravelling (by topoisomerases), during DNA synthesis, and during DNA repairs, and these breaks can be single- or double-stranded, (ii) breaks variously associated with unravelling, synthesis and repair can occur ‘anywhere, anytime’ (pre-synthesis, synthesis or post-synthesis) in the cell cycle, and (iii) the enzyme assemblies for DNA unravelling, synthesis and repair which make and religate the breaks must be non-covalently tethered to the ends of the DNA strands while the breaks created by the enzymes are in existence. It is then suggested that all the morphological types and other phenomena of chromosomal aberrations can be explained by aspects, mechanisms and effects of failures of this tethering function. Circumstances involving the basic mechanism (failure of DNA-end-tethering function while enzyme-created breaks are in existence) are described which might result in ‘gaps’, translocations (‘exchanges’), complex lesions such as ‘triradials’, as well as in ‘minutes’, amplifications and inversions. Predictions are made concerning likely results in various suggested studies including those involving sensitive assays for DNA-end-to-enzyme tethering functions in vitro.     

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.     

Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

DNA double strand breaks (DSBs) can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ). Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.     

The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited

In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.     

Human nucleotide excision repair in vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extract.

We searched for nucleotide excision repair in human cell-free extracts using two assays: damage-specific incision of DNA (the nicking assay) and damage-stimulated DNA synthesis (the repair synthesis assay). HeLa cell-free extract prepared by the method of Manley et al. (1980) has a weak nicking activity on UV irradiated DNA and the nicking is only slightly reduced when pyrimidine dimers are eliminated from the substrate by DNA photolyase. In contrast to the nicking assay, the extract gives a strong signal with UV irradiated substrate in the repair synthesis assay. The repair synthesis activity is ATP dependent and is reduced by about 50% by prior treatment of the substrate with DNA photolyase indicating that this fraction of repair synthesis is due to removal of pyrimidine dimers by nucleotide excision. Psoralen and cisplatin adducts which are known to be removed by nucleotide excision repair also elicited repair synthesis activity 5-10 fold above the background synthesis. When M13RF DNA containing a uniquely placed psoralen adduct was used in the reaction, complete repair was achieved in a fraction of molecules as evidenced by the restoration of psoralen inactivated KpnI restriction site. This activity is absent in xeroderma pigmentosum group A cells. We conclude that our cell-free extract contains the human nucleotide excision repair enzyme activity.