Biomaterial Database

Measurement of damage-specific DNA incision by nucleotide excision repair in vitro. 1994

P Calsou, and B Salles

Laboratoire de Pharmacologie et Toxicologie Fondamentales du CNRS, Toulouse-France.

We have devised a method to evaluate the capacity of mammalian cell extracts to incise damaged DNA in vitro. The assay uses damaged-plasmid DNA as a substrate for nucleotide excision repair by cell extracts. During this process, enzymatic incision of the damaged DNA is followed by DNA resynthesis. Under our assay conditions, the DNA synthesis stage of excision repair is prevented by limiting dNTP concentration and including the specific DNA polymerase inhibitor aphidicolin. Incisions are quantitatively detected by [alpha-32P]dAMP incorporation catalysed by the Klenow fragment of E. coli DNA pol I at nicked sites in plasmids purified from incision reactions. Lesion-specific incision is an ATP-dependent process; it was observed in plasmids modified with three different DNA damaging agents and damage-dependent incisions were abolished with extracts from xeroderma pigmentosum excision-repair deficient cell lines, indicating that this in vitro incision assay is dealing with true nucleotide excision repair.

UI	MeSH Term	Description	Entries
D009711	Nucleotides	The monomeric units from which DNA or RNA polymers are constructed. They consist of a purine or pyrimidine base, a pentose sugar, and a phosphate group. (From King & Stansfield, A Dictionary of Genetics, 4th ed)	Nucleotide
D010761	Phosphorus Radioisotopes	Unstable isotopes of phosphorus that decay or disintegrate emitting radiation. P atoms with atomic weights 28-34 except 31 are radioactive phosphorus isotopes.	Radioisotopes, Phosphorus
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D002460	Cell Line	Established cell cultures that have the potential to propagate indefinitely.	Cell Lines,Line, Cell,Lines, Cell
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
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
D004256	DNA Polymerase I	A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair.	DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004260	DNA Repair	The removal of DNA LESIONS and/or restoration of intact DNA strands without BASE PAIR MISMATCHES, intrastrand or interstrand crosslinks, or discontinuities in the DNA sugar-phosphate backbones.	DNA Damage Response
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man