Identification, characterization, and purification of DNA glycosylase/AP lyases by reductive crosslinking to 2'-deoxyribooligonucleotides containing specific base lesions.
This paper describes a reductive amination crosslinking protocol that facilitates identification and characterization of a class of DNA repair enzymes, DNA glycosylase/AP lyases, which are involved in base excision repair. This crosslinking technique has been used to identify enzymes in crude extracts and in partially purified enzyme preparations, to isolate proteins for sequencing, and to confirm the reaction mechanism of members of this enzyme family. Chemical reduction of the Schiff's base enzyme-substrate intermediate to a stable amine results in the formation of an irreversible covalent bond between the substrate lesion situated within a 2'-deoxyoligonucleotide and the repair enzyme. This complex can be detected by gel electrophoresis and can also be isolated and analyzed by amino acid sequencing.
Chheda
A D
AD
Department of Pathology, New York University Medical Center, New York, New York, 10016, USA.
Teebor
G W
GW
Cunningham
R P
RP
D000818:Animals; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000302:isolation & purification*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005821:Genetic Techniques; D006801:Humans; D008956:Models, Chemical; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000235:genetics; D051381:Rats; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase