NE AS A HEALING HUB · The role of nuclear pore and inner nuclear envelope proteins in the regulation of recombination and repair pathways in budding yeast
FP7 — People (Marie Curie Actions)
- Duration
- 2010-07-01 → 2012-06-30
- EU contribution
- €174,065
- Participants
- 1
- Scheme
- MC-IIF
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Results in brief
The role of nuclear pore and inner nuclear envelope proteins in the regulation of recombination and repair pathways in budding yeast
Without even citing the example of the nuclear power plant accident, human beings are facing the challenge of DNA damage by radiation. Although radiation creates a broad spectrum of damage, DNA double strand breaks (DSBs) are one of the most hazardous, as DSBs can lead to genomic translocations and loss of essential genetic information, ultimately provoking cell death or a risk of cancer. At the nascent DSB, a host of proteins are recruited sequentially to respond to the damage. We have shown that damaged DNA is recruited to the nuclear periphery and that relocation is important for DSB processing. During the course of break relocation, a subset of nuclear pore proteins and Mps3, an essential inner nuclear membrane protein, provide binding sites for the DSBs. However, little is known about the exact mechanism of relocation and how this affects repair. To elucidate the kinetics and genetic requirements of molecular steps during DSB relocation and processing, we introduced a site-specific DSB and followed the position by quantitative fluorescence microscopy. We found that the phosphorylation of histone H2A S129 and following SWR1-dependent incorporation of H2A.Z are required for the relocation of DSB to the nuclear periphery. To test the chromatin positioning property of Htz1, we exploited a protein targeting assay. We found that targeting of Htz1 to the chromatin is sufficient to shift the locus to the nuclear periphery through the binding with Mps3 without the assistance of SWR1 complex. Recently, it has been shown that there is a locus-specific increase in the mobility of an induced DSB in yeast nuclei. Interestingly, the increased mobility was abolished in SWR1-deficient mutants and htz1 deletion mutant. Using chromatin immunoprecipitation assays, we showed that SWR1 complex is required for the binding of DSBs to both nuclear pores and Mps3, whereas a closely related remodeler INO80 complex was necessary only for the binding to Mps3. INO80 complex was not required for the DSB relocation to the nuclear periphery suggesting the binding between DSB and Mps3 is not essential for the DSB relocation. Similarly, although it has been reported that end resection, DNA damage checkpoint proteins, repair and telomerase machinery all play roles in the anchoring of DSBs to Mps3, we found that these are not required for the DSB relocation to the nuclear periphery. Taken together, we propose that both the recruitment of SWR1 complex and the eventual incorporation of Htz1 near the DSB increase the mobility of the locus and relocate the locus to the nuclear pores. DSBs may undergo further regulation by proteins including INO80 complex at the nuclear pore; in some cases the DSB may subsequently interact with Mps3 where it is sequestered from promiscuous recombination. Contact details: Chihiro Horigome, Ph.D. Susan M. Gasser's lab Friedrich Miescher Institute for Biomedical Research Maulbeerstrasse 66, CH-4058 Basel, Switzerland tel. (office): +41-(0)61-697-3501 e-mail: chihiro.horigome@fmi.ch
Data: CORDIS, © European Union
Project objective
DNA double strand breaks (DSBs) and telomeres both represent ends of a linear chromosome, and both can lead to dangerous genomic rearrangements. Recent studies in budding yeast suggest that persistent DSBs associate with factors in two perinuclear compartments, at nuclear pores and in the inner nuclear membrane (INM). The stability of short telomeres also involves telomere binding to an INM protein, Mps3, and unequal sister chromatid exchange in the rDNA array is prevented by binding another INM protein, Heh1. In the absence of this anchor, rDNA rings are excised leading to premature senescence. From these studies it is clear that the interaction of chromatin with the nuclear periphery contributes to genome stability, yet many questions remain concerning the relocalization and its functional consequences. Are these interactions part of a common mechanism to stabilize or process breaks? Do they cross-talk with each other or do the anchorage sites provide distinct activities specific to the context of the DSB? This proposal will examine the role of nuclear pore and INM proteins in telomere maintenance, rDNA stability and DSB repair, but comparing the outcome of damage at the three sites in various mutant strains. I will use high resolution fluorescence imaging to observe the movement of a randomly distributed DSB to the INM or to pores, which can be distinguished by use of fluorescent tagging and a nup133 mutation. I will determine which repair pathways are regulated by the pores and by INM factors involved in perinuclear anchoring, and finally will investigate whether common mechanisms regulate DSBs processing and the repression of recombination at telomeres or within the rDNA. Intruiguingly all of the nuclear envelope components implicated in this process have homologues in higher eukaryotic cells, which suggests that these mechanisms may be conserved.
Original text from CORDIS.
Participants
- Novartis Forschungsstiftung · BASELCoordinatorCity levelSwitzerland
Links
Data: CORDIS, © European Union