Hsp104-Dependent Remodeling of Prion Complexes Mediates Protein-Only Inheritance

Inheritance of phenotypic traits depends on two key events: replication of the determinant of that trait and partitioning of these copies between mother and daughter cells. Although these processes are well understood for nucleic acid–based genes, the mechanisms by which protein-only or prion-based genetic elements direct phenotypic inheritance are poorly understood. Here, we report a process crucial for inheritance of the Saccharomyces cerevisiae prion [PSI(+)], a self-replicating conformer of the Sup35 protein. By tightly controlling expression of a Sup35-GFP fusion, we directly observe remodeling of existing Sup35([PSI+]) complexes in vivo. This dynamic change in Sup35([PSI+]) is lost when the molecular chaperone Hsp104, a factor essential for propagation of all yeast prions, is functionally impaired. The loss of Sup35([PSI+]) remodeling by Hsp104 decreases the mobility of these complexes in the cytosol, creates a segregation bias that limits their transmission to daughter cells, and consequently diminishes the efficiency of conversion of newly made Sup35 to the prion form. Our observations resolve several seemingly conflicting reports on the mechanism of Hsp104 action and point to a single Hsp104-dependent event in prion propagation

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present

Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps

Immuno-gene therapy with interferon-beta before surgical debulking delays recurrence and improves survival in a murine model of malignant mesothelioma.

OBJECTIVES: Immuno-gene therapy of mesothelioma with an adenovirus encoding interferon-beta mediated strong antitumor responses in murine models with low but not high tumor burden. Our goals were to determine the mechanisms responsible for this loss of efficacy and to test the hypothesis that the combination of preoperative adenovirus encoding interferon-beta and surgical resection would be effective in treating bulky tumors. METHODS: Flank tumors of a mouse mesothelioma cell line were treated with adenovirus encoding interferon-beta or adenoviral vector encoding the bacterial protein beta-galactosidase. Cytotoxic T lymphocytes and tumor infiltration by T lymphocytes were measured. Tumors were surgically excised 72 hours later and tumor cells were injected in the contralateral flank to create a model of a metastatic focus. Tumor-free survival and distant metastatic disease were assessed. RESULTS: Immuno-gene therapy effectively treated small tumors (\u3c200 \u3emm(3)) but did not reduce the size of large (\u3e800 mm(3)) flank tumors. Although treatment with adenovirus encoding interferon-beta resulted in the generation of tumor-neutralizing splenocytes in large tumors, the number of T cells visualized within the tumors was minimal. Tumors treated with adenovirus encoding interferon-beta (versus adenoviral vector encoding the bacterial protein beta-galactosidase or phosphate-buffered saline solution) prior to debulking increased long-term tumor-free survival and resulted in two- to sixfold smaller foci of implanted tumor cells at 2 weeks postoperatively. CONCLUSIONS: The use of adenovirus encoding interferon-beta or surgical debulking alone is ineffective in treating large tumors, but combining preoperative adenovirus encoding interferon-beta and surgical debulking significantly reduces tumor recurrence and improves long-term tumor-free survival. We postulate that adenovirus encoding interferon-beta amplifies the cytotoxic T-lymphocyte antitumor response, allowing elimination of residual tumor cells

Immuno-gene therapy with interferon-beta before surgical debulking delays recurrence and improves survival in a murine model of malignant mesothelioma.

OBJECTIVES: Immuno-gene therapy of mesothelioma with an adenovirus encoding interferon-beta mediated strong antitumor responses in murine models with low but not high tumor burden. Our goals were to determine the mechanisms responsible for this loss of efficacy and to test the hypothesis that the combination of preoperative adenovirus encoding interferon-beta and surgical resection would be effective in treating bulky tumors. METHODS: Flank tumors of a mouse mesothelioma cell line were treated with adenovirus encoding interferon-beta or adenoviral vector encoding the bacterial protein beta-galactosidase. Cytotoxic T lymphocytes and tumor infiltration by T lymphocytes were measured. Tumors were surgically excised 72 hours later and tumor cells were injected in the contralateral flank to create a model of a metastatic focus. Tumor-free survival and distant metastatic disease were assessed. RESULTS: Immuno-gene therapy effectively treated small tumors (\u3c200 \u3emm(3)) but did not reduce the size of large (\u3e800 mm(3)) flank tumors. Although treatment with adenovirus encoding interferon-beta resulted in the generation of tumor-neutralizing splenocytes in large tumors, the number of T cells visualized within the tumors was minimal. Tumors treated with adenovirus encoding interferon-beta (versus adenoviral vector encoding the bacterial protein beta-galactosidase or phosphate-buffered saline solution) prior to debulking increased long-term tumor-free survival and resulted in two- to sixfold smaller foci of implanted tumor cells at 2 weeks postoperatively. CONCLUSIONS: The use of adenovirus encoding interferon-beta or surgical debulking alone is ineffective in treating large tumors, but combining preoperative adenovirus encoding interferon-beta and surgical debulking significantly reduces tumor recurrence and improves long-term tumor-free survival. We postulate that adenovirus encoding interferon-beta amplifies the cytotoxic T-lymphocyte antitumor response, allowing elimination of residual tumor cells