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.