The dynamics of yeast telomeres and silencing proteins through the cell cycle

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

Adeno-associated virus (AAV) is a small DNA virus belonging to the family of the parvoviridae. AAV infects the human population, however no disease has been associated with this virus. On the contrary, AAV was reported to have oncosuppressive activities. The AAV genome contains two genes: the Rep gene encoding four non-structural proteins (Rep78, 68, 52 and 40) and the Cap gene encoding the three capsid proteins. Rep78 is implicated in transcription, replication and site-specific integration of the viral DNA. It has been shown to block the cell cycle in G1, G2 and S phase with a complete inhibition of DNA synthesis. To be able to induce such a strong cell cycle arrest, Rep78 affects several proteins implicated in the regulation of the cell cycle, such as the family of the cell division cycle 25 (Cdc25) phosphatases. Rep78 has been shown to inactivate Cdc25A by binding to it and preventing it from accessing its substrates. Cdc25B shares conserved domains with Cdc25A and moreover has an important role in cell cycle regulation, especially in giving the signal for entry into mitosis. Thus, in this work, the interaction between Cdc25B and Rep78 was examined in more detail. Rep78 was found to bind to Cdc25B and to alter its intracellular localisation. Cdc25B is nuclear, but has to migrate into the cytoplasm at the end of the G2 phase to activate proteins needed for the entry into mitosis. The expression of Rep78, which is nuclear, leads to the sequestration of Cdc25B in the nucleus. Moreover, Rep78 by interacting with Cdc25B not only changes its localisation, but also inhibits its phosphatase activity. Our model is that Rep78 interacts with Cdc25B and thus retains it in the nucleus. This sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis, thus contributing to the G2/M arrest induced by Rep78. Moreover, the inhibition of Cdc25B phosphatase activity by Rep78 may also contribute to the cell cycle block. Rep78 also interacts with a number of cellular proteins implicated in DNA replication, DNA repair or recombination. Interactions between Rep78 and mini-chromosome maintenance 3 (MCM3), DNA polymerases α and δ, proliferating cell nuclear antigen (PCNA), Rad50 and Rad51 were identified in this work as well as possible interactions between Rep78 and Ataxia telangiectasia mutated (ATM) or Ataxia telangiectasia and Rad3 related (ATR) proteins. These interactions may contribute to AAV genome replication as well as its integration into the host DNA.

EPFL2009

The positioning and dynamics of origins of replication in the budding yeast nucleus

Thierry Laroche

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

2001

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast

Thierry Laroche

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

1999

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

EPFL2009