Abstract
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.
Highlights
Tution experiments with protein preparations from human cells have identified six core factors that are required for recognition and excision of bulky DNA modifications: XPA protein, XPC HR23B complex, replication protein A heterotrimer, multisubunit transcription factor IIH complex, and two structure-specific nucleases, XPG and XPF-ERCC1 [3]
We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t1⁄2 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
To further verify the importance of NER in the removal of chromium-DNA damage, we measured the rate of repair of chromium adducts in NERϩ WT/SV fibroblasts and two NERϪ cell lines that are deficient in other NER proteins, XPC and XPF (Fig. 1, B and C)
Summary
Tution experiments with protein preparations from human cells have identified six core factors that are required for recognition and excision of bulky DNA modifications: XPA protein, XPC HR23B complex, replication protein A heterotrimer, multisubunit transcription factor IIH complex, and two structure-specific nucleases, XPG and XPF-ERCC1 [3]. NER consists of two subpathways that specialize in the removal of DNA lesions from either the entire genome (global NER) or the transcribed strands (transcription-coupled NER) [2]. The initial damage recognition in global NER is usually performed by the XPC-HR23B complex [4, 5], which may require additional factors for sensing DNA modifications that cause relatively minor duplex distortions. Alkylation of DNA phosphates generates alkylphosphotriesters that, in Escherichia coli, are repaired via a non-NER process involving the Ada protein [13].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
