The Role Of Mre11 Nuclease Domain Iii In Dna Replication

The MRE11/RAD50/NBS1 complex (Mre11 complex) is a central player in most aspects of the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining, telomere maintenance and DNA damage checkpoint activation. And there are also evidences showed Mre11 complex regulate S phase checkpoint and prevent DSBs accumulation during chromosome replication.Here, we studied the function of Mre11 and Mre11 nuclease domain III in S phase. We found that Mre11 deficiencies by small RNA interference in human cell lines lead to hypersensitivities to S phase inhibitors Mitomycin (MMC), Hydroxyurea (Hu) and Aphidicolin (Aph) and to a delayed S phase progression. Mre11-deficient cells also show an increased histone H2AX phosphorylation signal throughout S phase and acquire more DSBs occurring at inverted repeats during DNA replication when transfected with a replicative plasmid carrying a 47bp inverted repeats, moreover, Mre11-deficient cells also promote cell cycle checkpoint protein kinases Chk1 active. The sensitivity to S phase inhibitors, the duration of S phase, the intensity of H2AX signal and the accumulation of DSBs were recovered to comparable control level when rescue Mre11 deficiencies with pMre11 rescue. Furthermore, the function of nuclease motif subset is controversial. We found that the sensitivity to S phase inhibitors, the duration of S phase, H2AX phosphorylation and DSBs occurring at inverted repeats in Mre11 nuclease domain III H129N mutation cells were greatly increased.Taken together, these data demonstrate that Mre11 deficiency and its nuclease domain III mutation acquires more DSBs damage signal arising from normal DNA replication, suggesting that loss of Mre11 and its nuclease domain III contributes to the spontaneously genomic instability and disease phenotypes observed in ATLD patients.