Deficient nucleotide excision repair activity in protein extracts from normal human lymphocytes.
DNA repair activity in human peripheral blood lymphocytes (PBL) has been investigated by various techniques. Here, we report the use of an in vitro assay in order to assess nucleotide excision repair activity (NER). The mechanism of this major repair process relies on two broad steps: first, recognition, incision and excision of the damaged DNA; second, repair synthesis on the gapped DNA. Briefly, damaged plasmids were incubated with whole cell extracts which allows one to quantify DNA repair synthesis. When NER was determined on plasmid DNA damaged with UV-light or cisplatin, PBL extracts showed no repair synthesis for unstimulated lymphocytes. Using a new in vitro assay measuring only the damage-specific DNA incision activity in cell extracts, we found that the incision step in the repair reaction was blocked in unstimulated PBL. By mixing PBL with XP (group A, B, C, D) extracts, no restoration of NER activity was observed. In addition, these lymphocytes also lacked DNA replication activity as determined with pre-incised plasmid substrate. However, a phytohemagglutinin treatment of PBL led to an extent of repair synthesis similar to that observed with extracts from lymphoblastoid cells. When lymphocytes were incubated in 20% serum medium with and without phytohemagglutinin, the repair activity increased dramatically after 24 h. During the activation of lymphocytes, the extent of repair synthesis was proportional to the percentage of cells in S phase of the cell cycle. Our results suggest that the blockage of the cell cycle in G0/G1 in PBL may be responsible for their lack of NER activity.
Barret
J M
JM
Laboratoire de Pharmacologie et Toxicologie Fondamentales, CNRS, Toulouse, France.
Calsou
P
P
Salles
B
B
D001798:Blood Proteins / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D004247:DNA / Q000097:blood; D004260:DNA Repair; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D009711:Nucleotides / Q000235:genetics* / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D013268:Stimulation, Chemical
D001798:Blood Proteins; D002457:Cell Extracts; D009711:Nucleotides; D010835:Phytohemagglutinins; D004247:DNA