Generation of single-nucleotide repair patches following excision of uracil residues from DNA.
Open Access
- 1 April 1992
- journal article
- Published by Informa UK Limited in Molecular and Cellular Biology
- Vol. 12 (4), 1605-1612
- https://doi.org/10.1128/mcb.12.4.1605
Abstract
The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.Keywords
This publication has 54 references indexed in Scilit:
- Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesisMutation Research/DNA Repair, 1991
- Animal cell DNA polymerases in DNA repairMutation Research/DNA Repair, 1990
- The enzymology of apurinic/apyrimidinic endonucleasesMutation Research/DNA Repair, 1990
- Structures and functions of DNA glycosylasesMutation Research/DNA Repair, 1990
- In vitro correction of G o T mispairs to G o C pairs in nuclear extracts from human cellsNature, 1989
- Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonucleaseBiochemistry, 1989
- Properties of the Main Endonuclease Specific for Apurinic Sites of Escherichia coli (Endonuclease VI)European Journal of Biochemistry, 1978
- Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase IJournal of Molecular Biology, 1977
- Repair replication in mammalian cells after X-irradiationMutation Research, 1972
- Endonuclease II of Escherichia coli. Degradation of partially depurinated deoxyribonucleic acidBiochemistry, 1971