Abstracts Category : Other

The role of cut repeat proteins in DNA repair and resistance to treatments

by Simran Kaur

The three CUT domains within CUX1 were shown to interact with the base excision repair (BER) enzyme, 8-oxoguanine DNA glycosylase (OGG1), and stimulate its glycosylase and AP/lyase enzymatic activities. These activities process the removal of the oxidized base lesion 8-oxoguanine and the subsequent strand incision step. We showed that Cux1-/- mouse embryonic fibroblasts proliferate poorly in hyperoxia (20% oxygen). In addition, we showed that CUT domains accelerate DNA repair of oxidized bases and are recruited to DNA damage sites in cells, underscoring the role of CUX1 in oxidative stress. We also showed that the CUX1 paralog CUX2 interacts with OGG1 and stimulates its enzymatic activities. We extended these findings to the protein SATB1, which contains two CUT domains. 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 OGG1 cleavage activity in cell extracts. In parallel, we observed an increase in phospho-CHK1 and -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 eGFP-SATB1 fusion protein was rapidly recruited to laser microirradiation-induced DNA damage. Using purified proteins, we showed that SATB1 interacts directly with OGG1, increases OGG1 binding to 8-oxoguanine containing DNA, promotes Schiff base formation and stimulates OGG1 glycosylase and AP/lyase enzymatic activities. Structure/function analysis demonstrated that CUT domains, but not the homeodomain, are responsible for the stimulation of OGG1 activity. Using in vitro incision assays with purified proteins, we established that the CUT domains also stimulate the activity of the second enzyme in the BER pathway, apurinic/apyrimidinic endonuclease 1 (APE1). In agreement with these findings, CUX1 knockdown causes an increase in the number of abasic sites in genomic DNA and a decrease in APE1 activity as measured in cell extracts. Conversely, ectopic CUX1 expression increases APE1 activity and lowers the number of abasic sites in genomic DNA. CUX1 resides on 7q22.1, the most frequently and highly amplified chromosomal region in glioblastomas. Using immunohistochemistry, we showed that CUX1 proteins are highly expressed in a tumor microarray of 150 glioblastoma samples. The resistance of glioblastoma cells to the mono-alkylating agent temozolomide is determined to some extent by the activity of APE1. The effect of CUX1 knockdown and overexpression on the resistance of glioblastoma cell lines to temozolomide was investigated. We found that CUX1 knockdown decreases, whereas CUX1 overexpression increases, the resistance of glioblastoma cells to temozolomide. Moreover, a short protein containing only two CUT domains, which is active in DNA repair but devoid of transcriptional activity, is still able to increase the resistance to temozolomide. These findings indicate that CUX1 expression level impacts on the response of glioblastomaAdvisors/Committee Members: Alain Nepveu (Supervisor).