ELG1 protein stabilizes in response to treatment with various DNA damaging agents. (A) In HEK 293T cells, the level of ELG1 was induced in a time dependent manner in response to DNA damage (10 Gy IR, 0.01% MMS for one hour, 4 mM hydroxyurea (HU) for three hours, or 0.4 nM aphidicolin (Aph) for two hours). o/n represents overnight. (B) mRNA level of ELG1 was not changed significantly after IR treatment. (C) The mRNA level of ELG1 was not significantly changed during cell cycle. (D) Treatment with 20 μM of a proteosomal inhibitor MG132 for three hours stabilized ELG1. (E) Ectopically expressing FLAG-ELG1 was stabilized by either MMS or UV treatment. no represents no treatment of DNA damaging agents. It should be pointed out that right after treatment the level of ELG1 did not show much difference compared to no treatment control.

ELG1 accumulates at the stalled DNA replication forks in response to DNA damage. (A) ELG1 made foci in response to different types of DNA damage. RPE cells stably expressing ELG1-CFP made distinct foci structures in the nucleus after treatment with DNA damaging agents. Pictures were taken after twelve hours recovery from treatment of DNA damaging agents (15 J/m² UV, 0.4 nM aphidicolin (Aph) for two hours or 0.01% MMS for one hour, or 1 mM hydroxyurea (HU) for overnight). (B) ELG1 forms foci post 12 hours in response to 0.01% MMS treatment. There was no significance overlap between ELG1 and γ-H2AX foci after MMS treatment. (C) Foci formation of ELG1 upon DNA damage is specific in S phase where cyclin A is expressed. (D) The foci of CFP-ELG1 were localized at the stalled replication sites. RPE cells expressing CFP-ELG1 were briefly labeled with BrdU after one-hour treatment of 0.01% MMS treatment. CFP-ELG1 and BrdU were detected using anti-CFP and anti-BrdU antibodies, respectively.

Reduced expression of ELG1 caused spontaneous DNA damage and increased sensitivity to MMS. (A) The reduction of ELG1 mRNA level was confirmed in several cell lines by RT-PCR. (B) Silencing of ELG1 by two different shRNAs (targeting N-terminus and upstream of the P loop) caused spontaneous DNA damage. Western blot analysis using polyclonal ELG1 antibodies confirmed the silencing of ELG1 expression in stable knockdown cells. The enhancement of DNA damage and the activation of checkpoints were confirmed by the increased phosphorylation of H2AX(γ-H2AX), ATM (p1981-ATM) and SMC1 (pSMC1) and the stabilization of p53 protein. Tubulin was used as an internal control. (C) Reduced expression of ELG1 caused MMS sensitivity in HCT116. (D) Silencing the expression of ELG1 enhanced spontaneous foci formation of γ-H2AX and p1981-ATM. The average number of foci observed in a cell from these cell lines is presented in a graph. At least 50 cells were counted in each cell line. (E) Silencing the expression of ELG1 by siRNA in RPE cells enhanced 53BP1 foci. Left panel is an example and right panel shows average number of foci in a cell counted from 50 different cells. Chromosome end-to-end fusion (F) and inversion (G) were observed in cell lines expressing low level of ELG1.

The reduction of ELG1 expression caused chromosome instability upon MMS treatment. Example of chromosomes in metaphase from control (HCT116 with an empty shRNA vector) and a cell line that expresses low level of ELG1 in (A) and HEK 293T cells transfected with control (CTRL) siRNA or siRNA specifically targeting ELG1 in (C) after 0.01% MMS treatment for one hour followed with twenty-four hours of recovery. (B and D) Number of cells having different number of breaks observed in experiment in (A and C) were counted from 40 cells in each cell line. The significance of difference (p value indicated on the graph) was determined by the t-test.

Reduced level of ELG1 reduced DSB-induced homologous recombination (HR) frequency whereas enhanced spontaneous HR frequency. (A) HR frequency in response to a DSB induced by I-SceI, was reduced when ELG1 was silenced by siRNA. Top panel shows the DNA structure for the HR assay. (B) Silencing of ELG1 enhanced spontaneous HR. Top panel shows the structure of the plasmid pBHRF before and after HR. Bottom panel shows a graph with the mean of two independent experiments. (C) Silencing of ELG1 expression enhanced sister chromatid exchange rate.

Chronic exposure to DNA damage caused reduction in ELG1 level in mRNA expression dependent manner. (A) Chronic exposure of cells with 0.01% MMS promoted ELG1 degradation. (B) The mRNA level of ELG1 was reduced during chronic exposure to 0.01% MMS.