Oligonucleotides Of Defined Sequence Research Articles

Chemical and enzymatic modifications of 5-methylcytosine at the intersection of DNA damage, repair, and epigenetic reprogramming.

The DNA of all living organisms is persistently damaged by endogenous reactions including deamination and oxidation. Such damage, if not repaired correctly, can result in mutations that drive tumor development. In addition to chemical damage, recent studies have established that DNA bases can be enzymatically modified, generating many of the same modified bases. Irrespective of the mechanism of formation, modified bases can alter DNA-protein interactions and therefore modulate epigenetic control of gene transcription. The simultaneous presence of both chemically and enzymatically modified bases in DNA suggests a potential intersection, or collision, between DNA repair and epigenetic reprogramming. In this paper, we have prepared defined sequence oligonucleotides containing the complete set of oxidized and deaminated bases that could arise from 5-methylcytosine. We have probed these substrates with human glycosylases implicated in DNA repair and epigenetic reprogramming. New observations reported here include: SMUG1 excises 5-carboxyuracil (5caU) when paired with A or G. Both TDG and MBD4 cleave 5-formyluracil and 5caU when mispaired with G. Further, TDG not only removes 5-formylcytosine and 5-carboxycytosine when paired with G, but also when mispaired with A. Surprisingly, 5caU is one of the best substrates for human TDG, SMUG1 and MBD4, and a much better substrate than T. The data presented here introduces some unexpected findings that pose new questions on the interactions between endogenous DNA damage, repair, and epigenetic reprogramming pathways.

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Oxidative base damage to DNA: specificity of base excision repair enzymes.

Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago.

Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects.

Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT. This confirms that Tg is an excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS. From these data it may be suggested that 5-OHC could be modified during its enzymatic excision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.

DNA Colony Hybridization Method Using Synthetic Oligonucleotides to Detect Enterotoxigenic Escherichia coli: Collaborative Study

The genes that encode several of the enterotoxins produced by Escherichia coli have been cloned by recombinant DNA techniques. When the nucleotide sequence of these genes is determined, defined sequence oligonucleotides that include a part of these genes may be synthesized. A 22-base DNA hybridization probe was produced for each of 2 heat-stable E. coli enterotoxin (ST) genes: STH, from strains originally isolated from humans; and STP, from strains first found in pigs. For this study, 32P end-labeled DNA probes, sonicated calf thymus DNA, and 3 known and 20 unknown (10 ST-positive and 10 ST-negative) strains were sent to each of 23 collaborators. Cultures were spotted onto an agar-based medium and grown into colonies, which were transferred by blotting to cellulose filters, lysed by alkali and steam, and used for DNA colony hybridization with the ST DNA probes. Strains containing an ST gene were recognized as dark spots on an autoradiogram. Of the 460 samples analyzed, 440 (95.7%) were correctly classified by the collaborators. The method has been adopted official first action.

ChemInform Abstract: POLYMER SUPPORT SYNTHESIS. X. AN IMPROVED SYRINGE TECHNIQUE FOR THE PREPARATION OF OLIGONUCLEOTIDES OF DEFINED SEQUENCE

Chemischer InformationsdienstVolume 15, Issue 8 Natural Products ChemInform Abstract: POLYMER SUPPORT SYNTHESIS. X. AN IMPROVED SYRINGE TECHNIQUE FOR THE PREPARATION OF OLIGONUCLEOTIDES OF DEFINED SEQUENCE H. SELIGER, H. SELIGERSearch for more papers by this authorC. SCALFI, C. SCALFISearch for more papers by this authorF. EISENBEISS, F. EISENBEISSSearch for more papers by this author H. SELIGER, H. SELIGERSearch for more papers by this authorC. SCALFI, C. SCALFISearch for more papers by this authorF. EISENBEISS, F. EISENBEISSSearch for more papers by this author First published: February 21, 1984 https://doi.org/10.1002/chin.198408301AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume15, Issue8February 21, 1984 RelatedInformation

The use of barium salts of protected deoxyribonucleoside-3' p-chlorophenyl phosphates for construction of oligonucleotides by the phosphotriester method: high-yield synthesis of dinucleotide blocks.

A new experimental approach to the synthesis of polydeoxyribonucleotides via the phosphotriester method involves construction of oligonucleotide blocks by direct use of the easily prepared barium salts of O5',N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates as the key monomers in condensation reactions. The procedure has been demonstrated by the rapid synthesis in high yield and purity of all sixteen fuly protected dinucleotides (Formula: see text) (where dN' = dT, dbzC, dbzA, or dibG; (Formula: see text) This set of molecules constitutes a "syllabary" for the preparation of defined sequence oligonucleotides.