Michel Charbonneau

Cdc13 Cooperates with the Yeast Ku Proteins and Stn1 To Regulate Telomerase Recruitment

ABSTRACT The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both onSTN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

Fertilization of amphibian eggs: a comparison of electrical responses between anurans and urodeles.

In Pleurodeles waltl and Ambystoma mexicanum, which exhibit physiological polyspermy, the membrane potential in most eggs did not change in any consistent pattern during 45 min after fertilization; in some cases, a slow hyperpolarization began 5 to 15 min after insemination and continued for 10-15 min. These eggs then slowly depolarized, reaching a stable value of -10 to +10 mV, about 45 min after fertilization. Membranes of eggs activated by A23187 or by electrical stimulus showed a similar behavior. The diversity of responses does not correlate with the number of sperm fusing with the egg. Holding the membrane potential at a constant value between -40 and +40 mV during insemination did not prevent fertilization nor delay sperm-egg interactions. The fertilization or activation potential of Rana temporaria eggs consists of a rapid (1 sec) depolarization accompanied by a sudden decrease in membrane resistance. The activation potential can be triggered by A23187 and by calcium iontophoresis; its amplitude depends on the (Cl-)0 and to a lesser extent on the (Na+)0. Fertilization was prevented when the membrane potential was clamped above +15 mV. However, slowing the rise time (5 to 8 sec instead of 1 sec) and reducing the amplitude (10-20 mV instead of 40-60 mV) of the fertilization potential, both by injecting negative current, never induced polyspermy.

A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms.

Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.

Early Events in Anuran Amphibian Fertilization: An Ultrastructural Study of Changes Occurring in the Course of Monospermic Fertilization and Artificial Activation

In unfertilized frog eggs, the plasma membrane displays an animal vegetal polarity characterized by the presence of short microvilli in the vegetal hemisphere and long microvilli or ridge‐like protrusions in the animal hemisphere. The densities of microvilli are similar in the two hemispheres.

Anuran fertilization: a morphological reinvestigation of some early events

The morphological events dealing with the fertilization or activation process of the eggs of different Rana species are reported. Steps from sperm penetration to male pronucleus formation are described as well as the egg surface evolution until the second polar body formation. Depending on the species, a “fertilization body” (FB) rapidly differentiates at the site of sperm entry. This FB is either extruded or is reincorporated into the egg. In both cases a landmark persists. For the first time, it is clearly demonstrated with SEM that the cortical-granule exocytosis wave starts from the site of sperm entry. Just after the exocytosis wave occurs, the villi elongate and the movements of the pigment granules increase, reflecting a change in the egg cortex fluidity. This last phenomenon should be considered as new criteria of activation. The cell coat appears just as the cortical granule material is extruded; it is no longer possible to recognize the new membranes.

The Rad51 Pathway of Telomerase-Independent Maintenance of Telomeres Can Amplify TG1-3 Sequences in yku and cdc13 Mutants of Saccharomyces cerevisiae

ABSTRACT In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70Δ mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70Δ cells survived telomere deprotection by exclusively amplifying TG1-3 repeats (type II recombination). In a background lacking telomerase (tlc1Δ), this process predominated over type I recombination (amplification of subtelomeric Y′ sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70Δ or yku70Δ tlc1Δ mutants, but also in cdc13-1 YKU70+ tlc1Δ mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70Δ rad51Δ and cdc13-1 tlc1Δ rad51Δ cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG1-3 sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.

Intracellular pH and the increase in protein synthesis accompanying activation of Xenopus eggs

Metabolic activation following egg fertilization corresponds to an increase in protein synthesis and the initiation of DNA synthesis, which lead to cell division and development of the embryo. Since in several biological systems protein synthesis is regulated by intracellular pH (pHi), we have decided to investigate the situation during Xenopus egg activation. We confirmed that egg activation is accompanied by a pHi rise of 0.3 pH unit. Measurements of the rates of protein synthesis in unactivated and activated eggs, after microinjection of 3H‐leucine, demonstrated that activation was followed by a 2.5‐fold increase. Treatment of unactivated eggs with weak bases also increased pHi, but did not result in an increase in the rate of protein synthesis. Moreover, in vitro translation in cytoplasmic extracts was found to be pH‐independent, at least between 6.8 and 8.2.

Mitotic cyclins regulate telomeric recombination in telomerase-deficient yeast cells

ABSTRACT Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways. The Rad51 pathway amplifies the subtelomeric Y′ sequences, while the Rad50 pathway amplifies the telomeric TG1-3 repeats. Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate. The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway. We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors. This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2. In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed. This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function. The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres. They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.

Telomerase- and Rad52-Independent Immortalization of Budding Yeast by an Inherited-Long-Telomere Pathway of Telomeric Repeat Amplification

ABSTRACT In the absence of telomerase, telomeres erode, provoking accumulation of DNA damage and death by senescence. Rare survivors arise, however, due to Rad52-based amplification of telomeric sequences by homologous recombination. The present study reveals that in budding yeast cells, postsenescence survival relying on amplification of the TG1-3 telomeric repeats can take place in the absence of Rad52 when overelongated telomeres are present during senescence (hence its designation ILT, for inherited-long-telomere, pathway). By growth competition, the Rad52-independent pathway was almost as efficient as the Rad51- and Rad52-dependent pathway that predominates in telomerase-negative cells. The ILT pathway could also be triggered by increased telomerase accessibility before telomerase removal, combined with loss of telomere protection, indicating that prior accumulation of recombination proteins was not required. The ILT pathway was dependent on Rad50 and Mre11 but not on the Rad51 recombinase and Rad59, thus making it distinct from both the type II (budding yeast ALT [alternative lengthening of telomeres]) and type I pathways amplifying the TG1-3 repeats and subtelomeric sequences, respectively. The ILT pathway also required the Rad1 endonuclease and Elg1, a replication factor C (RFC)-like complex subunit, but not Rad24 or Ctf18 (two subunits of two other RFC-like complexes), the Dnl4 ligase, Yku70, or Nej1. Possible mechanisms for this Rad52-independent pathway of telomeric repeat amplification are discussed. The effects of inherited long telomeres on Rad52-dependent recombination are also reported.

Weak bases inhibit cleavage and embryogenesis in amphibians and echinoderms.

The action of weak bases was studied on the early embryonic development of a number of species. Gastrulation was disrupted in the frog, Xenopus laevis, the newt, Pleurodeles watlii, the sea urchins, Paracentrotus lividus and Sphaerechinus granularis and the starfish, Asterias rubens. This required only submillimolar amounts of either NH+4 (pH 9.0) or procaine (pH 8.2). At higher concentrations even early cell division was inhibited in all the species with furrow regression particularly noticeable in Xenopus eggs. A similar action of the weak bases on early development, the lack of any action at lower extracellular pH, and the counteracting action of NH+4 on acidity-induced disruption of sea urchin development, all implicate an elevation of intracellular pH. However, a more direct intracellular action of the weak bases cannot be ruled out.