DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

Abstract

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid. Applying the same dose of IR to Prdkc −/− and Ku −/− mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc −/− cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku −/− MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-016-0590-9) contains supplementary material, which is available to authorized users.

Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes

“…Prior work has shown that Sertoli cells are reliant on the c-NHEJ pathway for DSB repair [35, 38]. Results here suggest that DSB repair is compromised in the Nono gt /0 Sertoli cells in vivo .…”

“…Prior work has shown that 53BP1 is a more specific marker of DNA damage in the testes than γ-H2AX, which accumulates at programmed DSBs associated with meiosis in developing germ cells [35, 36]. The kinetics of 53BP1 foci formation have been characterized in cultured wild type and c-NHEJ deficient Sertoli cells.…”

“…The kinetics of 53BP1 foci formation have been characterized in cultured wild type and c-NHEJ deficient Sertoli cells. As in other cell types, a 4 h post-irradiation timepoint provides good discrimination between genotypes [35]. …”

“…The effects of DNA damaging agents on the testes have been well characterized, and other DNA repair deficiencies often lead to a testis phenotype [26, 27]. Prior work has also shown that NONO and other DBHS proteins are expressed in a cell-type specific pattern in the testes [28, 29] and, separately, that the c-NHEJ pathway is active in the testes and provides a major mechanism of protection from ionizing radiation 35. In the course of the work it also emerged that compensatory up-regulation of PSPC1 is not observed in the testes of NONO-deficient mice, reducing concerns over this as a potentially confounding factor.…”

SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining

“…Prior work in mammalian cells has shown that the number of 53BP1 foci reaches a maximum at 30 min post-irradiation and declines thereafter ( 48 – 50 ). The number of residual foci at 4 h post-irradiation is significantly greater in c-NHEJ deficient cells than in their wild-type counterparts ( 51 ).…”