DEVELOPMENT OF A NEW EASY COMPLEMENTATION ASSAY FOR DNA-REPAIR DEFICIENT HUMAN SYNDROMES USING CLONED REPAIR GENES

Nucleotide excision repair (NER)-deficient human cells have been assig ned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair gene s. We describe a new technique, based on the host cell reactivation as say, for the rapid determination of the complementation group of NER-d eficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and phot osensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a clo ned repair gene. Expression of the reporter gene, either chloramphenic ol acetyltransferase (CAT) or luciferase, reflects the DNA repair abil ity restored by the introduction of the appropriate repair gene. All g enetically characterized XP, CS and TTD/XP-D cells tested failed to ex press the UV-irradiated reporter gene, this reflecting their NER defec iency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme act ivity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI a nd XP19VI cells. Using this method, we assigned three new NER-deficien t human cells obtained from patients presenting clinical symptoms desc ribed as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of met hods now available to determine the complementation group of new NER d eficient patients with the advantage, unlike the somatic cell fusion a ssay or the microinjection procedure, of being simple, rapid, and inex pensive.