RECONSTITUTION OF THE DNA-BASE EXCISION-REPAIR PATHWAY

Background: The base excision-repair pathway is the major cellular def ence mechanism against spontaneous DNA damage. The enzymes involved ha ve been highly conserved during evolution. Base excision-repair has be en reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, w e have attempted to reconstitute the pathway in vitro using purified e nzymes. Results: We report here the successful reconstitution of the b ase excision-repair pathway with five purified enzymes from Escherichi a coil: uracil-DNA glycosylase, a representative of the DNA glycosylas es that remove various lesions from DNA; the AP endonuclease IV that s pecifically cleaves at abasic sites; RecJ protein which excises a 5' t erminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligas e. The reaction proceeds with high efficiency in the absence of additi onal factors in the reconstituted system. Four of the enzymes are abso lutely required for completion of the repair reaction. An unusual feat ure we have discovered is that the pathway branches after enzymatic in cision at an abasic DNA site. RecJ protein is required for the major r eaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, wit h generation-of longer repair patches by the 5' nuclease function of D NA polymerase I. Conclusions: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enz ymes. No additional protein factors seem to be required. There is a mi nor, back-up pathway that uses replication factors to generate longer repair patches.