Abstract
CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.
Highlights
CUX2 contains three Cut repeat domains and is expressed in postmitotic neurons
As a first step to investigate whether CUX2 plays a role in the repair of oxidative DNA damage, we tested whether the level of DNA damage would change in cortical neurons following the knockdown of Cux2
The drastic increase observed at pH 10 after treatment with formamidopyrimidine-DNA glycosylase indicates that the level of oxidized bases is increased when CUX2 expression is reduced (Fig. 1D)
Summary
Results: Cut repeats stimulate the OGG1 DNA glycosylase, and lower or higher CUX2 expression, respectively, delays or accelerates repair of oxidative DNA damage. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. The abbreviations used are: ROS, reactive oxygen species; BER, base excision repair; 8-oxoG, 7,8-dihydro-8-oxoguanine; AP, apurinic/apyrimidinic; CR, Cut repeat; MEF, mouse embryo fibroblast; HD, homeodomain; NLS, nuclear localization signal; 7-AAD, 7-aminoactinomycin D; OGG1, 8-oxoguanine DNA glycosylase 1; NTH1, Nth homolog 1; NEIL, Nei-like; HOXB3, homeodomain protein B3; hOGG1, human OGG1. Our results show that genetic inactivation or knockdown of Cux negatively affects the repair of oxidative DNA damage, whereas ectopic expression of various recombinant proteins that contain CUX2 Cut repeats can accelerate DNA repair. We show that HCC38 breast tumor cells are acutely dependent for their survival on the DNA repair activities of CUX2, thereby revealing another case of non-oncogene addiction involving a CUX protein
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