CUT Domain Proteins in DNA Repair and Cancer.

Recent studies revealed that CUT domains function as accessory factors that accelerate DNA repair by stimulating the enzymatic activities of the base excision repair enzymes OGG1, APE1, and DNA pol ß. Strikingly, the role of CUT domain proteins in DNA repair is exploited by cancer cells to facilitate their survival. Cancer cells in which the RAS pathway is activated produce an excess of reactive oxygen species (ROS) which, if not counterbalanced by increased production of antioxidants, causes sustained oxidative DNA damage and, ultimately, cell senescence. These cancer cells can adapt by increasing their capacity to repair oxidative DNA damage in part through elevated expression of CUT domain proteins such as CUX1, CUX2, or SATB1. In particular, CUX1 overexpression was shown to cooperate with RAS in the formation of mammary and lung tumors in mice. Conversely, knockdown of CUX1, CUX2, or SATB1 was found to be synthetic lethal in cancer cells exhibiting high ROS levels as a consequence of activating mutations in KRAS, HRAS, BRAF, or EGFR. Importantly, as a byproduct of their adaptation, cancer cells that overexpress CUT domain proteins exhibit increased resistance to genotoxic treatments such as ionizing radiation, temozolomide, and cisplatin.

CUT Domains Stimulate Pol ß Enzymatic Activities to Accelerate Completion of Base Excision Repair.

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.

Special AT-rich Sequence-binding Protein 1 (SATB1) Functions as an Accessory Factor in Base Excision Repair.

Base excision repair is initiated by DNA glycosylases that recognize specific altered bases. DNA glycosylases for oxidized bases carry both a glycosylase activity that removes the faulty base and an apyrimidinic/apurinic lyase activity that introduces a single-strand DNA incision. In particular, the CUT domains within the CUX1 and CUX2 proteins were recently shown to interact with the 8-oxoguanine (8-oxoG) DNA glycosylase and stimulate its enzymatic activities. SATB1, which contains two CUT domains, was originally characterized as a T cell-specific genome organizer whose aberrant overexpression in breast cancer can promote tumor progression. Here we investigated the involvement of SATB1 in DNA repair. SATB1 knockdown caused a delay in DNA repair following exposure to H2O2, an increase in OGG1-sensitive oxidized bases within genomic DNA, and a decrease in 8-oxoG cleavage activity in cell extracts. In parallel, we observed an increase in phospho-CHK1 and γ-H2AX levels and a decrease in DNA synthesis. Conversely, ectopic expression of SATB1 accelerated DNA repair and reduced the levels of oxidized bases in genomic DNA. Moreover, an enhanced GFP-SATB1 fusion protein was rapidly recruited to laser microirradiation-induced DNA damage. Using purified proteins, we showed that SATB1 interacts directly with OGG1, increases its binding to 8-oxoG-containing DNA, promotes Schiff base formation, and stimulates its glycosylase and apyrimidinic/apurinic lyase enzymatic activities. Structure/function analysis demonstrated that CUT domains, but not the homeodomain, are responsible for the stimulation of OGG1. Together, these results identify another CUT domain protein that functions both as a transcription factor and an accessory factor in base excision repair.