Point mutation at the Nbs1 Threonine 278 site does not affect mouse development, but compromises the Chk2 and Smc1 phosphorylation after DNA damage

NBS1, mutated in Nijmegen breakage syndrome (NBS), senses the DNA double strand breaks (DSBs) and initiates the DNA damage response (DDR) by activating ATM kinase. Meanwhile, NBS1 is phosphorylated by ATM at Serine 278 and Serine 343 and thereby assists the activation of the ATM downstream targets. To study the physiological function of the Nbs1 phosphorylation, we have knocked in a point mutation in the moue genome that results in the replacement of Threonine 278 (equivalent to the human Serine 278) by Alanine. The Nbs1T278A knock-in mice develop normally and show no gross defects. The mutation of this phosphorylation site does not affect the proliferation or genomic stability. Ionizing radiation (IR) of primary Nbs1T278A MEFs reveals no obvious defects in the Chk2 phosphorylation at 1 Gy, but a delayed phosphorylation of Chk2 and Smc1 only at intermediate (4.5 Gy) and high (10 Gy) doses, respectively. In contrast to Serine 343 mutant, Threonine 278 mutation has no effect on the HU-induced ATR-Chk1 activation. Our study thus shows that Nbs1 phosphorylation at the Threonine 278 is dispensable for mouse development and plays a differential function in assisting the DDR of downstream effectors in vivo, depending on the doses of DNA damage.

► Generation of knock-in mice carrying a point mutation in Threonine 278 of Nbs1.
► Nbs1-T278 mutant mice develop normally.
► Nbs1 Threonine 278 plays a fine-tuning function in response to DNA damage.