Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity.
Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.
Reynolds
Mindy
M
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912, USA.
Peterson
Elizabeth
E
Quievryn
George
G
Zhitkovich
Anatoly
A
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002840:Chromates / Q000633:toxicity*; D002857:Chromium / Q000737:chemistry* / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016877:Oxidants / Q000494:pharmacology; D010710:Phosphates; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein
D002840:Chromates; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D016877:Oxidants; D010710:Phosphates; D034741:RNA, Small Interfering; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D002857:Chromium; D004247:DNA; D006861:Hydrogen Peroxide