Abstract
BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.
Highlights
Loss of genomic integrity is a hallmark for tumorigenesis
We demonstrate that BCCIP deficiency causes the formation of a unique type of structural abnormality of chromosomes called sister chromatid union (SCU)
We have found that BCCIP deficiency causes chromatid abnormalities especially a dramatic induction of sister chromatid unions (SCUs), and impairs mouse embryo development
Summary
Mammalian cells maintain genomic integrity by ensuring DNA replication fidelity in S-phase, equal chromosome distribution into daughter cells during mitosis, error-free repair of sporadic DNA damage throughout the cell cycle, and a coordinated cell cycle progression [1]. Homologous recombination (HR) plays roles in repair of DNA double strand breaks (DSB) and in replication fidelity [2,3]. When the replication forks stall during Sphase, one-ended DSBs are produced on one of the sister chromatids at the stalled replication fork. The HR machinery uses the 39-end of a single-stranded tail of the oneended DSB to invade the intact double-stranded DNA at the collapsed replication fork, which leads to the resolution of the stalled fork. Replication stress is a status highly susceptible to genomic instability
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