Reconstitution of the DNA base excision-repair pathway.
BACKGROUND
The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.


RESULTS
We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I.


CONCLUSIONS
Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.
Dianov
G
G
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Lindahl
T
T
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D045647:DNA Glycosylases; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases; D008190:Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C489467:RecJ protein, E coli; C070765:recJ protein, Bacteria; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases