Phototriggered Formation and Repair of DNA Containing a Site-Specific Single Strand Break of the Type Produced by Ionizing Radiation or AP Lyase Activity
| ISSN: | 1520-4995 0006-2960 |
|---|---|
| DOI: | 10.1021/bi001781j |
| Přístupová URL adresa: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7b75b582b9fc6ac0ae1495d20e23ef2f https://doi.org/10.1021/bi001781j |
| Přírůstkové číslo: | edsair.doi.dedup.....7b75b582b9fc6ac0ae1495d20e23ef2f |
| Autor: | Kaijiang Zhang, John-Stephen Taylor |
| Rok vydání: | 2000 |
| Předmět: |
Exonuclease
Polynucleotide 5'-Hydroxyl-Kinase DNA Repair Photochemistry Ultraviolet Rays Carbon-Oxygen Lyases Genetic Vectors DNA Single-Stranded Biochemistry chemistry.chemical_compound DNA-(Apurinic or Apyrimidinic Site) Lyase Bacteriophage T4 Nucleotide Phosphorylation chemistry.chemical_classification Exonuclease III DNA ligase biology Escherichia coli Proteins Nucleic Acid Heteroduplexes DNA DNA Polymerase I Deoxyribonuclease IV (Phage T4-Induced) Enzyme Activation A-site Oligodeoxyribonucleotides chemistry Duplex (building) Mutagenesis Site-Directed biology.protein Biophysics DNA polymerase I DNA Damage |
| Zdroj: | Biochemistry. 40:153-159 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi001781j |
| Popis: | DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and multiply damaged sites that might otherwise be difficult to prepare by other methods due to their lability. |
| Databáze: | OpenAIRE |
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