Abstract
The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.
Highlights
In vitro and in intact cells [5,6,7,8,9], presumably reflecting the limited access of nucleotide excision repair (NER) proteins to these lesions [10]
Overexpression of HMGA1 Proteins Inhibits NER in MCF7 Cells— Mammalian cells that are deficient in various aspects of DNA repair exhibit, in most cases, increased sensitivity to DNA-damaging agents [43]
To gain insight into whether the HMGA1 proteins play a role in NER of UV-induced cyclobutane pyrimidine dimers (CPDs) lesions in vivo, the UV sensitivity of genetically engineered human cell lines was examined by two methods, colony-forming ability and dye exclusion after UV exposure
Summary
In vitro and in intact cells [5,6,7,8,9], presumably reflecting the limited access of NER proteins to these lesions [10]. CPDs alter DNA structure from normal B-form, causing severe bending of the helical axis and disruption of Watson-Crick base pairs at the lesion sites [19] These lesion-induced distortions can obstruct progression of both RNA and DNA polymerases [20, 21]. HMGA1a is a proto-oncogene [31] that is overexpressed in a large number of different naturally occurring cancers [25] and whose experimental up-regulation induces metastatic progression and increased malignancy of neoplastic cells [32] Consideration of these characteristics of CPDs and HMGA proteins led us to suspect that these proteins might participate in the process of DNA repair or DNA damage formation at AT-rich sequences. The results of these studies demonstrated that HMGA1 overexpression in human cells inhibits their ability to survive exposure to UV light and significantly reduces their efficiency for global genomic nucleotide excision repair
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