Biomaterial Database

Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides. 2001

K J Bowman, and R L Pla, and Y Guichard, and P B Farmer, and G D Jones

Department of Oncology and Biomonitoring and Molecular Interactions Section, MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

UI	MeSH Term	Description	Entries
D009838	Oligodeoxyribonucleotides	A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D010084	Oxidation-Reduction	A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471).	Redox,Oxidation Reduction
D010727	Phosphoric Diester Hydrolases	A class of enzymes that catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. EC 3.1.4.	Phosphodiesterase,Phosphodiesterases,Hydrolases, Phosphoric Diester
D010755	Organophosphates	Carbon-containing phosphoric acid derivatives. Included under this heading are compounds that have CARBON atoms bound to one or more OXYGEN atoms of the P(	Organophosphate,Phosphates, Organic,Phosphoric Acid Esters,Organopyrophosphates,Acid Esters, Phosphoric,Esters, Phosphoric Acid,Organic Phosphates
D002851	Chromatography, High Pressure Liquid	Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed.	Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004249	DNA Damage	Injuries to DNA that introduce deviations from its normal, intact structure and which may, if left unrepaired, result in a MUTATION or a block of DNA REPLICATION. These deviations may be caused by physical or chemical agents and occur by natural or unnatural, introduced circumstances. They include the introduction of illegitimate bases during replication or by deamination or other modification of bases; the loss of a base from the DNA backbone leaving an abasic site; single-strand breaks; double strand breaks; and intrastrand (PYRIMIDINE DIMERS) or interstrand crosslinking. Damage can often be repaired (DNA REPAIR). If the damage is extensive, it can induce APOPTOSIS.	DNA Injury,DNA Lesion,DNA Lesions,Genotoxic Stress,Stress, Genotoxic,Injury, DNA,DNA Injuries
D005663	Furans	Compounds with a 5-membered ring of four carbons and an oxygen. They are aromatic heterocycles. The reduced form is tetrahydrofuran.	Tetrahydrofurans
D000478	Alkylation	The covalent bonding of an alkyl group to an organic compound. It can occur by a simple addition reaction or by substitution of another functional group.	Alkylations
D013941	Thymine	One of four constituent bases of DNA.	5-Methyluracil,5 Methyluracil
D043264	Phosphodiesterase I	A phosphoric diester hydrolase that removes 5'-nucleotides from the 3'-hydroxy termini of 3'-hydroxy-terminated OLIGONUCLEOTIDES. It has low activity towards POLYNUCLEOTIDES and the presence of 3'-phosphate terminus on the substrate may inhibit hydrolysis.	5'-Exonuclease,5'-Nucleotidephosphodiesterase,Alkaline Phosphodiesterase,Alkaline Phosphodiesterase I,Oligonucleate 5'-Nucleotidohydrolase,Orthophosphoric Diester Phosphohydrolase,5' Exonuclease,5' Nucleotidephosphodiesterase,Diester Phosphohydrolase, Orthophosphoric,Oligonucleate 5' Nucleotidohydrolase,Phosphodiesterase I, Alkaline,Phosphodiesterase, Alkaline,Phosphohydrolase, Orthophosphoric Diester