A role for p53 in DNA end rejoining by human cell extracts.
The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.
Bill
C A
CA
Department of Cancer Biology, Harvard University School of Public Health, Boston, MA 02115, USA.
Yu
Y
Y
Miselis
N R
NR
Little
J B
JB
Nickoloff
J A
JA
D002457:Cell Extracts; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D002457:Cell Extracts; D016159:Tumor Suppressor Protein p53