RECONSTITUTION OF HUMAN DNA-REPAIR EXCISION NUCLEASE IN A HIGHLY DEFINED SYSTEM

Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free e xtracts indicate that at least 16 polypeptides are required to carry o ut the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of t hese five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the exc ised fragment remains associated with the post-incision DNA-protein co mplex, suggesting that accessory proteins are needed to release the ex cised oligomer.