Our results demonstrate that ELG1 is a new mammalian DNA damage response protein involved in DNA repair following exposure to various genotoxic stresses during DNA replication. Higher levels of spontaneous DNA damage (), higher sensitivity to DNA damaging agents () and higher persistent DSBs even twenty-four hours following exposure to DNA damaging agent () when the expression of ELG1 was silenced strongly argue that ELG1 plays a critical role in repairing DNA damage. Protein stabilization and foci formation by DNA damage in S phase ( and ) suggest that ELG1 functions in DNA repair during DNA replication. Because ELG1 accumulates at DNA replication stall sites (), we propose that ELG1 functions at stalled sites of DNA replication. Because ELG1 forms an RLC complex,8
the ELG1 RLC might load or unload PCNA or ubiquitinated PCNA from chromatin to facilitate DNA repair. However, recent in vitro biochemical analysis of yeast Elg1p failed to detect PCNA loading or unloading activity.24
The absence of ELG1 leads to the accumulation of spontaneous DNA damage (). ELG1 therefore have a physiological role in repairing spontaneous DNA damage despite the fact that the level of ELG1 without DNA damage is relatively low. Constant oxidative stress and DNA sequences that could stall DNA replication such as repetitive sequences could be sources for spontaneous DNA damage requiring ELG1 for repair. In addition, DNA damage from abnormal Okazaki fragment maturation in the lagging strand synthesis could be another source for spontaneous DNA damage requiring ELG1 for repair because yeast Elg1p interacts with Rad27p, a yeast homologue of human FEN1 flap endonuclease which specifically removes RNA primers during lagging strand synthesis.8
Consistently, ELG1 makes spontaneous as well as damage-induced S phase specific foci ( and data not shown).
Recently, Ishii et al. reported that the level of the mouse Elg1 (Frag1) mRNA is rapidly decreased upon chronic replication stress.13
We also observed that the chronic exposure of human cells to replication stress caused reduction in ELG1 mRNA and caused apoptosis ( and data not shown). In contrast, following treatment with and recovery from various DNA damaging agents, ELG1 protein levels were stabilized () and ELG1 formed nuclear foci () within six hours. Therefore, ELG1 responds to various forms of DNA damage that block DNA replication and facilitates DNA repair. However, if DNA damage is too high for cells to recover, ELG1 mRNA appears to be rapidly degraded and cell death occurs.
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.
I DSB induced HR was reduced by siRNA knockdown of ELG1 (). Similarly, the elg1
mutation in yeast generated reduced HR upon MMS or phleomycin treatment.21
In stark contrast to DSB-induced HR, increases in spontaneous HR and sister chromatid exchange were observed in ELG1 knockdown cells (). These results suggest that the lack of ELG1 enhances spontaneous recombinational repair. The higher HR frequency in the ELG1 knockdown cells indicates that low expression of ELG1 could increase more replication associated DSBs that initiate HR. Consistently, there was high increase of γ-H2AX and 53BP1 foci in the ELG1 knockdown cells (). Similarly, the yeast elg1
mutant exhibited a thirty-fold increase in the level of long terminal repeat recombination, a five-fold increase in ectopic recombination between Ty elements and a five-fold elevation of direct repeat recombination.7
These phenotypes of the yeast elg1
mutant appeared to be caused by the increase of DNA damage during DNA replication and may indicate the same in human cells. ELG1 has a clear, but complex role in HR, which needs to be investigated further due to its complex role in multiple pathways affecting HR such as DNA replication, chromatin cohesion and DNA damage sensing.6–9,21,25–27
Collectively, our results illustrate a critical role for human ELG1 in the maintenance of genomic stability and suggest that ELG1 may be an important tumor suppressor. To date, no mutation in ELG1 has been directly linked to any cancer-prone syndrome. Intriguingly, high incidence of cancers in approximately 5–10% of Neurofibromatosis type I patients who have a micro-deletion of the NF1
gene together with several other genes, including ELG1
suggests a putative role of ELG1 in the suppression of carcino-genesis. Therefore, ELG1 could be a candidate as a new tumor suppressor gene.