DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.
Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation. Chromosomal integrity is essential for normal growth and development. In eukaryotes, there are three essential complexes of proteins that are central to chromosome dynamics. These are cohesin, condensin, and the Smc5/6 complex, known collectively as the structural maintenance of chromosomes (SMC) complex. Each complex shares a related architecture, and central to them are a heterodimer of SMC proteins: Smc1 and -3 in cohesin, Smc2 and -4 in condensin, and Smc5 and -6 in Smc5/6. These SMC proteins are large coiled-coil molecules with globular N and C termini containing Walker A and B ATP-binding motifs. They fold and interact at a flexible hinge, with ATP acting to hold the globular domains together. A kleisin protein bridges each heterodimer to form a putative ring-shaped structure, and each complex has specific additional non-SMC proteins that serve as regulators and effectors of function (1-3).Chromosome condensation and sister chromatid cohesion are the key roles for condensin and cohesin, respectively (1). However, defects in DNA repair have also been described for yeast strains harboring hypomorphic mutant alleles of condensin (4) and cohesin (5-7) subunits. In the case of cohesin, a role in DNA repair could stem from the fact that DNA repair by homologous recombination (HR) requires the sister chromatid to be in close proximity to the damaged chromatid. However, more recently cohesin in mammalian cells has been shown to also act as a transcriptional insulator in collaboration with CTCF (8, 9), and so the DNA repair function of cohesin may be more complex than a simple scaffolding mechanism. As its name suggests, deciphering Smc5/6 function has proved more elusive, though most studies have focused on a role for this complex in...
The lethality of eso1 mutants is due to activation of the spindle assembly checkpoint and is triggered by premature centromere separation. This novel role for Eso1 at the centromeres is independent of Smc3 acetylation, and the defect arises in an Smc5/6-dependent manner.
Chromosomes are subjected to massive re-engineering as they are replicated, transcribed, repaired, condensed and segregated into daughter cells. Among the engineers are three large protein complexes collectively known as the Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6. As their names suggest, cohesin controls sister chromatid cohesion, condensin controls chromosome condensation, and while precise functions for Smc5/6 have remained somewhat elusive, most reports have focused on the control of recombinational DNA repair. Here, we focus on cohesin and Smc5/6 function. It is becoming increasingly clear that the functional repertoires of these complexes is greater than sister chromatid cohesion and recombination. These SMC complexes are emerging as inter-related and cooperating factors that control chromosome dynamics throughout interphase. However, they also release their embrace of sister chromatids to enable their segregation at anaphase, resetting the dynamic cycle of SMC-chromosome interactions.
AIM:To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes. METHODS:Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured. RESULTS:Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore, the concentration of MDA in group E100 at 36 h was significantly higher by 4.5-and 1.7-fold, respectively, than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group. CONCLUSION:These results suggest that longtime incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation.
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