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Nucleic Acids Research, 1994, Vol. 22, No. 23 4937-4942
© 1994

MOLECULAR BIOLOGY

Properties of damage-dependent DNA incision by nucleotide excision repair in human cell-free extracts

Patrick Calsou* and Bernard Salles

Laboratoire de Pharmacologie et Toxicologie Fondamentales du CNRS 205 route de Narbonne, 31077 Toulouse Cedex, France

*To whom correpsondence should be addressed

Received September 6, 1994. Revised October 17, 1994. Accepted October 17, 1994.

Nucleotide excision repair (NER) is the primary mechanism for the removal of many lesions from DNA. This repair process can be broadly divided in two stages: first, incision at damaged sites and second, synthesis of new DNA to replace the oligonucleotide removed by excision. In order to dissect the repair mechanism, we have recently devised a method to analyze the incision reaction in vitro in the absence of repair synthesis (1). Damage-specific incisions take place in a repair reaction in which mammalian cell-free extracts are mixed with undamaged and damaged plasmids. Most of the incision events are accompanied by excision. Using this assay, we investigated here various parameters that specifically affect the level of damage-dependent incision activity by cell-free extracts in vitro. We have defined optimal conditions for the reaction and determined the kinetics of the incision with cell-free extracts from human cells. We present direct evidence that the incision step of NER is ATPdependent. In addition, we observe that Mn2+ but no other divalent cation can substitute for Mg2+ in the incision reaction.


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