Biomedical subjects

Geoffrey M Kapler

Publications and source records attributed to Geoffrey M Kapler.

4 recordsLinked to original sources

PubMed2006

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.

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PubMed2005

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.

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PubMed2004

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.

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PubMed2003

Characterization of a novel origin recognition complex-like complex: implications for DNA recognition, cell cycle control, and locus-specific gene amplification.

The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

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