Giovanni Lucchini

Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase.

The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.

Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae.

Abstract. Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pds1 is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mps1 protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Esp1 separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2Δ cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.

Initiation of protein synthesis in isolated mitochondria and chloroplasts

N5-Formyltetrahydrofolate, a competitive inhibitor of the formylation of the initiator Met-tRNAfMet in an in vitro assay, is a powerful inhibitor of amino acid incorporation in isolated Saccharomyces cerevisiae mitochondria and in Euglena gracilis chloroplasts. Thus, a large part of the incorporation is dependent upon new initiation acts. On the contrary, the rate of incorporation can be largely increased by addition of the specific formyl group donor, N10-formyltetrahydrofolate. Experiments are also reported strongly suggesting that the formylation of Met-tRNAfMet is an absolute requirement in order to initiate protein synthesis in chloroplasts, as has been shown in mitochondria.

Endogenous synthesis of formyl-methionine peptides in isolated mitochondria and chloroplasts

Abstract In the presence of a system for the incorporation of aminoacids, formyl group donor and puromycin, mitochondria and chloroplasts can initiate “in vitro” the synthesis of peptides with formyl-methionine at the N-terminus. This indicates that in these organelles endogenous messenger(s) programme polypeptide chains starting with formyl-methionine.

Mn2+ and Mg2+ uptake in Mn-sensitive and Mn-resistant yeast strains

Abstract Mn2+ and Mg2+ uptake was studied in a wild type strain of S. cerevisiae and in a mutant resistant to Mn2+. Contrary to expectations, the resistant strain accumulates Mn2+ to a higher concentration than the wild type strain, while Mg2+ uptake is unaffected. Moreover, the transmembrane electric potential (PD) of the resistant strain is far less depolarized by Mn2+ than that of the sensitive strain. A possible explanation for these results is discussed.

Mutants resistant to manganese in Saccharomyces cerevisiae

SummarySeveral mutants resistant to Mn2+ have been isolated and characterized in Saccharomyces cerevisiae. All the mutations are semidominant and allelic to a single nuclear gene (MNRI). Mg2+ in the growth medium reverses the inhibitory effect of Mn2+ in a competitive way. This appears to be due to the inhibition of the uptake of Mn2+ by the cells, not to an increase of the amount of Mg2+ inside the cells.The analysis of the distribution of Mn2+ taken up by growing cells shows that the amount of the ion present in insoluble form is far higher in resistant than in sensitive cells. We therefore believe that yeast cells have a sequestering system for Mn2+ and that the major difference between mutants and wild-type strains lies in the much higher efficiency of this system.

Eukaryotic N10-formyl-H4folate:Methionyl-tRNAf transformylase: Some properties of the Euglena gracilis enzyme

Abstract The N 10 - formyl-H 4 folate:methionyl-tRNA fMet transformylase was extracted from Euglena cells, and the level of enzyme activity found to be of 0.15 pmol/min per 106 cells for autotrophic and 0.016 pmol/min per 106 cells for streptomycin-bleached cells. The enzyme has been purified by DEAE-cellulose and CM-Sephadex chromatography about 1000-fold from autotrophic cells and about 650-fold from chloroplasts obtained by the non-aqueous technique. A methionyl-tRNA synthetase with apparently high specificity for the initiator tRNAMet has also been found which allowed the estimation of enzyme activity even with unfractionated tRNAs from different sources, since only formylatable species were present in methionylated form. As judged from the initial velocity the Euglena transformylase showed the same kinetic behaviour towards Met-tRNAfMet from E. gracilis, Escherichia coli and yeast. The chloroplast enzyme showed different properties from those of the bacterial enzyme. It appears to have a partial requirement for K+, a higher molecular weight, and a lower Mg2+ optimum. Moreover, the chloroplast transformylase showed sigmoidal rather than hyperbolic kinetics respect to Met-tRNAfMet ( n H = 1.81 ; K′ = 4 · 10 −14 ) as well as N 10 - formyl-H 4 folate ( n H = 2.46 ; K′ = 5.2 · 10 −16 ), thus indicating a strong positive cooperativity for both substrates.

The yeast DNA polymerase-primase complex: genes and proteins.

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.

Control of glucose phosphorylation in Euglena gracilis. I. Partial characterization of a glucokinase.

Abstract In Euglena gracilis strain Z, grwon either under autotrophic or heterotrophic conditions, glucose and fructose phosphorylation occurs by means of two distinct enzymatic activities, which may be completely separated by centrifugation at 105 000 × g. Fructose is phosphorylated by a soluble fructokinase (ATP: d -fructose 6-phosphotransferase, EC 2.7.1.4) whereas a particulate glucokinase (ATP: d -glucose 6-phosphotransferase, EC 2.7.1.2), with high K m (8 mM) for glucose, is the only enzyme able to phosphorylate glucose. Kinetic analysis indicates that glucokinase is activated allosterically by orthophosphate. Activation by orthophosphate occurs, in vitro , in at least two ways: (1) by relieving inhibition by Mg-ATP complex; (2) by direct activation of the enzyme.