Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates.

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.

RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin.

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.

The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks.

Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Delta maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

Molecular mechanism of NFAT family proteins for differential regulation of the IL-2 and TNF-alpha promoters.

Nuclear factor of activated T cells (NFAT) is a family of transcription factors that regulates activation-induced transcription of many immunologically important genes. Although all NFAT family proteins contain a highly conserved DNA-binding domain and also bind cooperatively with AP-1 proteins to the interleukin-2 (IL-2) promoter NFAT site, each member shows characteristic site preferences to other promoters. Previously, we have shown that NFATc.beta, an isoform of NFATc, is different from NFATp in both DNA binding and transactivation of the TNF-alpha promoter. To further characterize target gene specificity of NFATc and NFATp, we generated deletion mutants as well as mutants swapping the C-terminal region of their DNA binding domains, and analyzed their DNA-binding specificity to different target sites. Our results show that the C-terminal one third of DNA binding domain confers different binding specificity of NFATc and NFATp to an NFAT site in the TNF-alpha promoters. Transient expression of the mutant NFAT proteins also demonstrates that transcriptional activation of the target promoters is consistent with the DNA binding specificity of the mutant NFATs. These results strongly suggest that a binding site preference and availability of different NFAT proteins may program the temporal expression of distinct cytokine genes. Importantly, the C-terminal region of the DNA binding domain plays an important role in determining the binding site preferences at least of NFATp and NFATc members.

RACK-1, a receptor for activated C kinase, interacts with the transcription factor NFAT and represses its transactivation.

To isolate and characterize a novel protein that interacts with nuclear factor of activated T cells (NFAT) and potentially regulates its activity, we screened a Jurkat cDNA library by using the NFAT regulatory domain as bait in the yeast two-hybrid system. RACK-1, a receptor for activated protein kinase C and a homologue of the G-protein beta subunit, was identified as a NFAT-binding protein. Mammalian two hybrid tests in CV-1 cells and a coimmunoprecipitation assay confirmed protein-protein interaction between NFAT and RACK-1. In addition, overexpression of RACK-1 specifically suppressed transcriptional activation derived by NFAT, but not by NF-kappaB. These results demonstrate RACK-1 as a potent negative modulator of NFAT activation and suggest a novel mechanism in NFAT regulation.

RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination.

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

In vitro selection of specific RNA inhibitors of NFATc.

NFAT (nuclear factor of activated T cells) plays a pivotal role in inducible gene transcription during the immune response and functions as a major target for immunosuppressive drugs such as cyclosporin A and FK-506. However, due to toxic effects of these drugs, which arise from their ability to inhibit calcineurin in non-immune cells, development of agents that directly target NFAT without toxic effects is warranted. Here, we present an in vitro selection of RNA aptamer to NFATc DNA binding domain (DBD) from a combinatorial RNA library with 41 nucleotide-long random sequences using the SELEX technique. The selected (SE) RNA was found to specifically and avidly bind NFATc DBD based on immunoprecipitation and competitive gel retardation assay. SE RNA also efficiently and specifically inhibited DNA binding capacity of NFATc, but not NFATp. Furthermore, transient RNA transfection studies show that only SE RNA can selectively and efficiently inhibit the NFATc- but neither the NFkappaB- nor NFATp-driven promoter activity in cells. These results suggest that SE RNA identified in this study is a specific inhibitor of NFATc activation, and hence, can be used not only for the study of NFAT functions but for the development of potent immune modulating agents.

Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

Retinoids are potent immune modulators that inhibit Fas ligand (FasL) expression and thereby repress the activation-induced apoptosis of immature thymocytes and T-cell hybridomas. In this study, we demonstrate that all-trans-retinoic acid (all-trans-RA) directly represses the transcriptional activity of the nuclear factors of activated T-cells (NFAT), which is an important transactivator of the FasL promoter. The analysis of reporter constructs containing the FasL promoter and wild-type or mutant NFAT binding-sites indicated that all-trans-RA repression was mediated via an NFAT binding element located in the promoter. A reporter construct comprising the NFAT binding sequence linked to a heterologous SV-40 promoter showed that NFAT transcriptional activity was significantly inhibited by all-trans-RA. Furthermore, all-trans-RA inhibited activation of the distal NFAT binding motif present in the interleukin (IL)-2 promoter, suggesting that the inhibition of NFAT function by all-trans-RA was not specific to the FasL promoter. Gel shift assays corroborated the results of the gene reporter studies by showing that all-trans-RA decreased the NFAT binding to DNA. All-trans-RA blocked translocation of NFATp from the cytosol into the nucleus, which was induced by PMA/ionomycin treatment in HeLa cells transfected with a Flag-tagged NFATp. Taken together, our results indicate that FasL inhibition by all-trans-RA involves a novel mechanism whereby the transcriptional function of NFAT is blocked.