Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins

Abstract

Previous work has established that stably-transfected human MCF7 cells over-expressing high mobility group A1 proteins (HMGA1) are deficient in global genomic repair (GGR) following exposure to either UV light or cisplatin. To investigate whether HMGA1 over-expression also interferes with gene-specific repair, we employed a rapid and convenient quantitative polymerase chain reaction assay for measuring repair in unique DNA sequences. Efficiency of UV-induced lesion removal was assessed for two genes in MCF7 cells either induced, or not, to over-express transgenic HMGA1 proteins: the constitutively active HPRT gene and the transcriptionally silent β-globin gene. As controls, similar experiments were also performed in non-transgenic MCF7 cells that do not express detectable levels of HMGA1 and in normal human embryonic fibroblasts that naturally overexpress HMGA1 proteins. Our results indicate that exposure of cells to a UV dose of 20 J/m 2 produced an average of 0.21 ± 0.03 lesions/kb and 0.19 ± 0.02 lesions/kb in the HPRT and β-globin genes, respectively, with no significant difference between HMGA1 over-expressing cells and nonexpressing cells. On the other hand, analysis of repair following UV exposure revealed that, compared to controls, HMGA1 over-expressing cells take considerably longer to repair photo-lesions in both the active HPRT and the silent β-globin loci, with non-expressing cells repairing 50% of lesions in HPRT 3-4 hours faster than HMGA1 over-expressing cells. Interestingly, the delay in repair is even more prolonged in the silent β-globin locus in HMGA1 over-expressing cells compared to control cells. To our knowledge, this is the first report of HMGA1 proteins inhibiting NER within specific genes located in either transcriptionally active "open", or inactive "closed", chromatin domains. Furthermore, taken together with previous findings, these results suggest that HMGA1 overexpression interferes with repair processes common to both the GGR and transcription coupled repair pathways.

HMGA1 Levels Influence Mitochondrial Function and Mitochondrial DNA Repair Efficiency

“…Interestingly, the differences in mitochondrial mass and mtDNA and ROS levels between the malignant Hs578Bst and normal Hs578T cells are either equal to, or greater than, the differences in these parameters between the transgenic OFF and ON cells. This disparity may be attributed to genetic differences between these cell types or may even be caused by factors other than the (2,81). At least two molecular mechanisms contribute to this NER deficiency: (i) inhibition by HMGA1 of the transcription of DNA repair genes (e.g., in xeroderma pigmentosum A) whose encoded protein products are required for NER to occur (2) and (ii) direct binding of HMGA1 to UV-induced DNA lesions [e.g., cyclobutane pyrimidine dimers and (6-4) photoproducts], which prevents access of repair enzymes to the lesions (2).…”

“…Details of the creation and use of the transgenic HA7C line of human MCF-7 breast adenocarcinoma cells containing a tetracycline-regulated hemagglutinin-taggedHMGA1a cDNA expression cassette have been reported previously (2,3,81,100,116). When grown for several days in medium without tetracycline, HA7C cells express high levels (elevated ϳ40-fold over basal levels) of the hemagglutinin-HMGA1a protein (hereinafter referred to as HMGA1) (117).…”

“…Methods for the quantitative evaluation of DNA repair efficiency employing long-range QPCR amplification have been described in detail elsewhere (81,105,106). Briefly, 25 l of each QPCR mixture was loaded onto a standard 0.7% agarose gel containing 2 M ethidium bromide, and the amplified DNA products were separated by electrophoresis.…”

“…Time course analyses of the rate of repair of oxidative damage in nearly full-length (16.2-kb) mtDNA fragments were performed using modifications of the long-range QPCR methods developed by Santos and his coworkers (105, 106) as described previously (81). Briefly, prior to QPCR amplification, approximately 2 g of purified total cellular DNA was digested to completion with the restriction enzyme XhoI (4 U; New England Biolabs) for 14 h at 37°C to linearize human mtDNA (GenBank accession no.…”

The HMGA proteins: A myriad of functions (Review)

“…It has been shown by Adair et al that HMGA1 overexpression in human cells inhibits their ability to survive exposure to relatively high doses of UV light and that it also significantly reduces their ability for general genomic nucleotide excision repair. One way in which HMGA1 can hamper nucleotide excision repair is by physically inhibiting access of DNA repair-associated proteins to CPD lesions, by directly binding to the UV-induced lesions (30,31). Likewise, HMGA1 proteins might negatively regulate transcription of certain genes that code for repair factors, as is shown for the nucleotide excision repair factors ERCC1 (32), and Xeroderma Pigmentosum Group A (XPA) (33).…”