BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

Aneuploidy and tumorigenesis

Aneuploidy is a prominent phenotype of cancer. It refers to deviations from the normal number of chromosomes in a cell, as a result of whole-chromosome loss or gain. In most cases, aneuploidy is caused by mitotic errors due to defects in the mechanisms that have evolved to ensure faithful chromosome segregation, such as the spindle assembly checkpoint (SAC). The observation that SAC-deficient mice are tumor prone demonstrates that this checkpoint is important in suppressing tumor formation and suggests that aneuploidy can induce tumorigenesis. In this review, we will summarize our current knowledge about the cause of aneuploidy and discuss the cellular response to aneuploidy in the context of tumorigenesis.     

Fed-batch fermentation of indole-3-acetic acid production in stirred tank fermenter by red yeast <Emphasis Type="Italic">Rhodosporidium paludigenum</Emphasis>

Indole-3-acetic acid (IAA) is a significant secondary metabolite that is the most important auxin of plant hormones. Production of IAA is considered to be a key trait to support plant growth. The improvement of IAA production by a basidiomycetous red yeast Rhodosporidium paludigenum DMKU-RP301 was investigated. Batch and fed-batch fermentation of R. paludigenum DMKU-RP301 were conducted in a 2 L stirred tank fermenter. Using batch fermentation, it was found that when cultivated at an agitation speed of 200 rpm and a 3 L/min aeration rate, this yeast produced IAA at its maximum level of 1,627.1 mg/L (9.7 mg/L/h). In fed-batch fermentation, a higher level of maximum IAA production than that found in batch fermentation was observed, i.e. 2,743.9 mg/L (25.4 mg/L/h). It is therefore suggested that fed-batch fermentation improves the efficiency of IAA production in terms of product concentration and IAA productivity. Moreover, yeast carotenoid production was also investigated using R. paludigenum DMKU-RP301, and found a maximum carotenoid production of 3.05 mg/L.     

Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast

Sister chromatid cohesion is normally established in S phase in a process that depends on the cohesion establishment factor Eco1, a conserved acetyltransferase. However, due to the lack of known in vivo substrates, how Eco1 regulates cohesion is not understood. Here we report that yeast Eco1 and its human ortholog, ESCO1, both acetylate Smc3, a component of the cohesin complex that physically holds the sister chromatid together, at two conserved lysine residues. Mutating these lysine residues to a nonacetylatable form leads to increased loss of sister chromatid cohesion and genome instability in both yeast and human. In addition, we clarified that the acetyltransferase activity of Eco1 is essential for its function. Our study thus identified a molecular target for the acetyltransferase Eco1 and revealed that Smc3 acetylation is a conserved mechanism in regulating sister chromatid cohesion.     

Early-onset aging and defective DNA damage response in Cdc14b-deficient mice

The Cdc14 dual-specificity phosphatase plays a key role in the mitotic exit of budding yeast cells. Mammals have two homologues, Cdc14a and Cdc14b. Unlike the yeast counterpart, neither Cdc14a nor Cdc14b seems to be essential for mitotic exit. To determine the physiological function of Cdc14b, we generated mice deficient in the phosphatase. The mutant mice were viable and did not display overt abnormalities. However, these mice developed signs of aging at much younger ages than the wild-type mice. At the cellular level, the Cdc14b-deficient mouse embryonic fibroblasts (MEFs) grew more slowly than the controls at later passages as a result of increased rates of senescence. Consistent with these premature-aging phenotypes, Cdc14b-deficient cells accumulated more endogenous DNA damage than the wild-type cells, and more Cdc14b-deficient MEFs entered senescence than control MEFs in response to exogenous DNA damage. However, no deficiencies in DNA damage checkpoint response were detected in Cdc14b mutant cells, suggesting that the function of Cdc14b is required for efficient DNA damage repair.     

Targeting of GFP-Cre to the Mouse Cyp11a1 Locus Both Drives Cre Recombinase Expression in Steroidogenic Cells and Permits Generation of Cyp11a1 Knock Out Mice

To permit conditional gene targeting of floxed alleles in steroidogenic cell-types we have generated a transgenic mouse line that expresses Cre Recombinase under the regulation of the endogenous Cytochrome P450 side chain cleavage enzyme (Cyp11a1) promoter. Mice Carrying the Cyp11a1-GC (GFP-Cre) allele express Cre Recombinase in fetal adrenal and testis, and adrenal cortex, testicular Leydig cells (and a small proportion of Sertoli cells), theca cells of the ovary, and the hindbrain in postnatal life. Circulating testosterone concentration is unchanged in Cyp11^+/GC males, suggesting steroidogenesis is unaffected by loss of one allele of Cyp11a1, mice are grossly normal, and Cre Recombinase functions to recombine floxed alleles of both a YFP reporter gene and the Androgen Receptor (AR) in steroidogenic cells of the testis, ovary, adrenal and hindbrain. Additionally, when bred to homozygosity (Cyp11a1^GC/GC), knock-in of GFP-Cre to the endogenous Cyp11a1 locus results in a novel mouse model lacking endogenous Cyp11a1 (P450-SCC) function. This unique dual-purpose model has utility both for those wishing to conditionally target genes within steroidogenic cell types and for studies requiring mice lacking endogenous steroid hormone production.     

Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand

A total of 1035 yeast isolates, obtained from rice and sugar cane leaves, were screened primarily for indole-3-acetic acid (IAA) production. Thirteen isolates were selected, due to their IAA production ranging from 1.2 to 29.3 mg g⁻¹ DCW. These isolates were investigated for their capabilities of calcium phosphate and ZnO₃ solubilisation, and also for production of NH₃, polyamine, and siderophore. Their 1-aminocyclopropane-1-carboxylate (ACC) deaminase, catalase and fungal cell wall-degrading enzyme activities were assessed. Their antagonism against rice fungal pathogens was also evaluated. Strain identification, based on molecular taxonomy, of the thirteen yeast isolates revealed that four yeast species – i.e. Hannaella sinensis (DMKU-RP45), Cryptococcus flavus (DMKU-RE12, DMKU-RE19, DMKU-RE67, and DMKU-RP128), Rhodosporidium paludigenum (DMKU-RP301) and Torulaspora globosa (DMKU-RP31) – were capable of high IAA production. Catalase activity was detected in all yeast strains tested. The yeast R. paludigenum DMKU-RP301 was the best IAA producer, yielding 29.3 mg g⁻¹ DCW, and showed the ability to produce NH₃ and siderophore. Different levels of IAA production (7.2–9.7 mg g⁻¹ DCW) were found in four strains of C. flavus DMKU-RE12, DMKU-RE19, and DMKU-RE67, which are rice leaf endophytes, and strain DMKU-RP128, which is a rice leaf epiphyte. NH₃ production and carboxymethyl cellulase (CMCase) activity was also detected in these four strains. Antagonism to fungal plant pathogens and production of antifungal volatile compounds were exhibited in T. globosa DMKU-RP31, as well as a moderate level of IAA production (4.9 mg g⁻¹ DCW). The overall results indicated that T. globosa DMKU-RP31 might be used in two ways: enhancing plant growth and acting as a biocontrol agent. In addition, four C. flavus were also found to be strains of interest for optimal IAA production.     

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.