Oligodeoxynucleotides as substrates for O/sup 6/-alkylguanine-DNA alkyltransferase (AGT)
AGT is a DNA repair protein which is known to catalyze the transfer of alkyl groups from the O/sup 6/-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. The authors have examined the ability of this protein isolated from both E. coli and mammalian cells to carry out this reaction using oligodeoxynucleotides containing O/sup 6/-methylguanine (m/sup 6/G). Dodecadeoxynucleotides of the sequence 5'-dCGBGAATTCm/sup 6/GCG-3' where B is any one of the normal 4 bases were all repaired very rapidly with 50% repair in less than 15 sec at 0/sup 0/C. The hexadeoxynucleotide 5'-dCGCm/sup 6/GCG-3' was repaired slightly more slowly with 50% removal requiring 7 min at 0/sup 0/C and 1.5 min at 37/sup 0/C. The tetradeoxynucleotide 5'-dTm/sup 6/GCA-3' was also a substrate for the protein but was repaired much more slowly requiring 45 min for 50% repair at 37/sup 0/C. These results indicate that (a) the AGT has a strong but not absolute preference for double stranded DNA substrates; (b) the repair of m/sup 6/G is independent of the base opposite the lesion; and (c) that very short oligodeoxynucleotides are substrates for repair by this protein. The latter property was used to set up a very sensitive assay procedure for the AGT. The 5' end of the 5'-dTm/sup 6/GCA-3' was labeled with /sup 32/P by use of polynucleotide kinase and the formation of (/sup 32/P)-5'-dTGACA-3' monitored by separating the methylated form from the unmethylated form by HPLC.
- Research Organization:
- Pennsylvania State Univ. College of Medicine, Hershey
- OSTI ID:
- 5097807
- Report Number(s):
- CONF-8606151-; TRN: 86-034801
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NUCLEOTIDES
DNA REPAIR
TRANSFERASES
ENZYME ACTIVITY
GUANINE
LIQUID COLUMN CHROMATOGRAPHY
PHOSPHORUS 32
SUBSTRATES
TRACER TECHNIQUES
AMINES
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PURINES
RADIOISOTOPES
RECOVERY
REPAIR
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques