Functional Dissection of P1 Bacteriophage Holin-like Proteins Reveals the Biological Sense of P1 Lytic System Complexity.

P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin-pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.

Cohesin Irr1/Scc3 is likely to influence transcription in Saccharomyces cerevisiae via interaction with Mediator complex.

The evolutionarily conserved proteins forming sister chromatid cohesion complex are also involved in the regulation of gene transcription. The participation of SA2p (mammalian ortholog of yeast Irr1p, associated with the core of the complex) in the regulation of transcription is already described. Here we analyzed microarray profiles of gene expression of a Saccharomyces cerevisiae irr1-1/IRR1 heterozygous diploid strain. We report that expression of 33 genes is affected by the presence of the mutated Irr1-1p and identify those genes. This supports the suggested role of Irr1p in the regulation of transcription. We also indicate that Irr1p may interact with elements of transcriptional coactivator Mediator.

[Sister chromatid cohesion complex in Eukaryota]. / Kompleks kohezyjny chromatyd siostrzanych u Eukaryota.

Faithful chromosome segregation in mitosis and meiosis requires the presence of the sister chromatid cohesion complex. The complex, which was initially identified and characterized in yeast Saccharomyces cerevisiae, and subsequently detected in other Eukaryota, is composed of four evolutionarily conserved core subunits (cohesins) Smc1, Smc3, Scc1/Mcd1 and Irr1/Scc3. Apart from the core proteins, accurate segregation requires also elements necessary for the deposition of cohesins and for the establishment and the regulation of cohesion. There are several models of cohesin structure and functioning. The oldest and the most popular ring model is currently replaced by the handcuff model. Regulation of cohesion is not very well established but the regulatory role of the Ecol, Irr1--STAG2, Pds5 and Wap1/Rad61 proteins seems undoubted. Meiotic cohesion differs from cohesion in mitosis and requires the specific Rec8 and Sgol proteins. Apart from the main function--the participation in chromosome segregation--cohesins are also involved in the regulation of transcription, DNA double-strand break repair and chromosome morphogenesis. Here we characterize elements of the complex, and describe models of the complex functioning. Moreover, we discuss the regulation of sister chromatid cohesion in mitosis and meiosis and, additionally, we describe atypical functions of cohesins.

The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle.

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.

Substitution F659G in the Irr1p/Scc3p cohesin influences the cell wall of Saccharomyces cerevisiae.

The sister chromatid cohesion complex of Saccharomyces cerevisiae is composed of proteins termed cohesins. The complex forms a ring structure that entraps sister DNAs, probably following replication. The mechanism of cohesion is universal and the proteins participating in this process are evolutionarily highly conserved. We investigated the Irr1p/Scc3p cohesin subunit, an under-studied protein. We show that the presence of a mutated copy of IRR1 gene, encoding the F658G substitution in Irr1p, changes the sensitivity of the heterozygous irr1-1/IRR1 diploid to cell wall-affecting compounds. Microscopic images indicate that chitin distribution in the mutant cell wall is affected, although the biochemical composition of the cell wall is not drastically changed. This observation suggests that irr1-1 mutation in heterozygous state may influence the cell wall integrity and indicates a possible link between mechanisms regulating the cell wall biosynthesis, nuclear migration and chromosome segregation.