Excision of nucleoside analogs in mitochondria by p53 protein.
OBJECTIVE
Nucleoside analogs, used against HIV, can be incorporated into a mitochondrial DNA by DNA polymerase gamma. Both the decrease in mitochondrial DNA and increased mutations of mitochondrial DNA may lead to mitochondrial diseases. The tumor suppressor protein p53 exhibits 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. In the present study, we investigated the ability of p53 to excise incorporated nucleoside analogs from DNA in mitochondria.


METHODS
The functional interaction of p53 and DNA polymerase gamma during the incorporation of nucleoside analog was examined in mitochondrial fractions of p53-null H1299 cells, as the source of DNA polymerase gamma.


METHODS
Primer extension reactions were carried out to elucidate the incorporation and removal of nucleoside analogs.


RESULTS
The results demonstrate that the excision of incorporated nucleoside analogs in mitochondrial fractions of H1299 cells increased in the presence of purified recombinant p53, or cytoplasmic extracts of large cell carcinoma 2 cells expressing endogenous wild-type p53 (but not specifically predepleted extracts) or cytoplasmic extracts of H1299 cells overexpressing wild-type p53, but not exonuclease-deficient mutant p53-R175H. The amount of nucleoside analogs incorporated into the elongated DNA with mitochondrial fractions of human colon carcinoma 116 (HCT116)(p53+/+) cells was lower than that of HCT116(p53-/-) cells. Furthermore, mitochondrion-localized elevation of p53 in HCT116(p53+/+) cells, following the irradiation-stress stimuli, correlates with the reduction in incorporation of nucleoside analogs and wrong nucleotides.


CONCLUSIONS
p53 in mitochondria may functionally interact with DNA polymerase gamma, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.
Bakhanashvili
Mary
M
Infectious Diseases Unit, Sheba Medical Center, Tel-Hashomer 52621, Israel. bakhanus@yahoo.com
Grinberg
Shai
S
Bonda
Elad
E
Rahav
Galia
G
D015153:Blotting, Western; D018287:Carcinoma, Large Cell / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004249:DNA Damage / Q000235:genetics*; D000074002:DNA Polymerase gamma; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D019286:Endopeptidase K; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1; D006801:Humans; D009705:Nucleosides / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D017931:DNA Primers; D004272:DNA, Mitochondrial; D004279:DNA, Viral; D009705:Nucleosides; D016159:Tumor Suppressor Protein p53; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D019286:Endopeptidase K