Current Biology
Research PaperReconstitution of the DNA base excision—repair pathway
Section snippets
Background:
Three different excision–repair pathways are universally present in living organisms, and constitute the main cellular strategies for removing lesions and pre-mutagenic errors from DNA. Strand-specific mismatch repair provides protection against occasional rare errors that occur during DNA replication and have escaped proof-reading mechanisms. Nucleotide excision–repair (NER) is responsible for correction of damage that causes major helix distortion, in particular the dipyrimidine adducts
Requirement for a DNA glycosylase in the in vitro reaction
Uracil residues in DNA can be generated by cytosine deamination and are repaired efficiently in vivo by a BER process initiated by uracil-DNA glycosylase [10]. Double-stranded oligonucleotides containing a centrally placed G.U base pair, surrounded by appropriate restriction enzyme sites, have been employed as substrates for in vitro reactions [4]. In previous work with E. coli extracts, the dUMP residue was replaced efficiently by a dCMP residue. No incorporation of newly synthesized material
Discussion
A general model for the BER process (Figure 6) may be proposed on the basis of the results described above and recent results obtained with cell extracts and partially purified components [4], [8]. This model is similar to previous schemes [18], [19], but the existence of a branched pathway and the events associated with removal of the deoxyribose-phosphate residue at an incised abasic site are new features. The main route, which has been reproduced with purified enzymes in the present study,
Reagent enzymes
The following E. coli enzymes were purified from enzyme-overproducing strains as described: uracil-DNA glycosylase [33], endonuclease IV [34], RecJ protein [13], [14] and Fpg protein [15]. E. coli DNA polymerase I, DNA ligase, exonuclease III and T4 polynucleotide kinase were obtained from Boehringer-Mannheim. Restriction endonuclease HpaII was purchased from New England Biolabs, Inc.
Cell extracts and oligonucleotide substrates
E. coli NH5033 (recB, sbcB, endA) was obtained from S.C. West, and E. coli BD10 (ung, thyA, deoC) and its ung+
Grigory Dianov and Tomas Lindahl (corresponding author), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK.
Present address for Grigory Dianov: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235,USA.
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Grigory Dianov and Tomas Lindahl (corresponding author), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK.
Present address for Grigory Dianov: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235,USA.