Ongoing DNA synthesis in the rat cerebral cortex is regulated by a proteolytic pathway independent of the proteasome and calpains.

By using mini-units of tissue and protease inhibitors in short term incubation (0-180 min), we studied the role of proteolysis for ongoing DNA replication in the developing rat cerebral cortex. The protease inhibitors TLCK, TPCK, PMSF, MG-132 and PSI markedly inhibited DNA synthesis. The inhibitory effects were concentration-dependent and of early onset (within 60 min). The most selective proteasome inhibitors lactacystin and clasto-lactacystin-beta-lactone as well as the calpain inhibitor I and II had no or minimal effects on DNA synthesis. Only high concentrations of calpain inhibitor I (>or= 250 microM) and calpain inhibitor II (>or= 500 microM) gave a DNA synthesis inhibition. These results suggest that (1) ongoing DNA replication is regulated by proteolysis and (2) the proteolytic pathways involved are neither the proteasome nor the calpains.

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena.

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.

TIF1 Represses rDNA replication initiation, but promotes normal S phase progression and chromosome transmission in Tetrahymena.

The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication. Despite this, replication of the rDNA minichromosome initiates precociously. Because rDNA copy number is unaffected in the polyploid macronucleus, mechanisms that prevent reinitiation appear intact. Although mutants exit macronuclear S with a wild-type DNA content, division of the amitotic macronucleus is both delayed and abnormal. Nuclear defects are also observed in the diploid mitotic micronucleus, as TIF1 mutants lose a significant fraction of their micronuclear DNA. Hence, TIF1 is required for the propagation and subsequent transmission of germline chromosomes. The broad phenotypes associated with a TIF1-deficiency suggest that this origin binding protein is required globally for the proper execution and/or monitoring of key chromosomal events during S phase and possibly at later stages of the cell cycle. We propose that micro- and macronuclear defects result from exiting the respective nuclear S phases with physically compromised chromosomes.

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila.

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.

Roscovitine inhibits ongoing DNA synthesis in human cervical cancer.

The effect of roscovitine, a purine analogue and cyclin dependent kinase inhibitor, on DNA synthesis rate in tissue mini-units obtained from human cervical cancers was investigated. Roscovitine (100 microM) gave a DNA synthesis rate inhibition by 61% (P<0.0001; range 23-93%) within 30 min of incubation. This inhibitory effect was concentration-dependent. The results suggest that the inhibition of tumor DNA synthesis rate is due to a direct effect on the DNA synthesis machinery via presently unknown mechanisms. In addition, the potential application of CDKs inhibitors as preventive agents is discussed.