The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

The late expression factors 8 and 9 and possibly the phosphoprotein p78/83 of Autographa californica multicapsid nucleopolyhedrovirus are components of the virus-induced RNA polymerase.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

A novel role for the budding yeast RAD9 checkpoint gene in DNA damage-dependent transcription.

Cells respond to DNA damage by arresting cell cycle progression and activating several DNA repair mechanisms. These responses allow damaged DNA to be repaired efficiently, thus ensuring the maintenance of genetic integrity. In the budding yeast, Saccharomyces cerevisiae, DNA damage leads both to activation of checkpoints at the G1, S and G2 phases of the cell cycle and to a transcriptional response. The G1 and G2 checkpoints have been shown previously to be under the control of the RAD9 gene. We show here that RAD9 is also required for the transcriptional response to DNA damage. Northern blot analysis demonstrated that RAD9 controls the DNA damage-specific induction of a large 'regulon' of repair, replication and recombination genes. This induction is cell-cycle independent as it was observed in asynchronous cultures and cells blocked in G1 or G2/M. RAD9-dependent induction was also observed from isolated damage responsive promoter elements in a lacZ reporter-based plasmid assay. RAD9 cells deficient in the transcriptional response were more sensitive to DNA damage than wild-type cells, even after functional substitution of checkpoints, suggesting that this activation may have an important role in DNA repair. Our findings parallel observations with the Escherichia coli SOS system and suggest the existence of an analogous eukaryotic network coordinating the cellular responses to DNA damage.

The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.

Identification and characterization of a baculovirus occlusion body glycoprotein which resembles spheroidin, an entomopoxvirus protein.

A 37-kDa polypeptide specified by Autographa californica nuclear polyhedrosis virus was found to share significant homology with Choristoneura biennis entomopoxyvirus spheroidin protein, which is the major component of entomopoxvirus occlusion bodies. Antibodies raised against spheroidin cross-reacted with the 37-kDa protein and confirmed its expression in the late phase of wild-type baculovirus infection. Immunoblot analysis and fluorescence microscopy demonstrated that the protein was associated with purified A. californica nuclear polyhedrosis virus occlusion bodies and was absent in purified virions. Immunofluorescence studies localized the protein to the periphery of occlusion bodies and the internal membranes of cells infected with wild-type baculovirus. The open reading frame encoding this spheroidinlike protein was inserted into a baculovirus expression vector, and recombinant protein was synthesized under control of the polyhedrin promoter. Studies of the recombinant protein demonstrated that it was heterogeneous in molecular mass as a result of N-linked glycosylation. Tunicamycin inhibited carbohydrate addition and yielded proteins of 34 and 33 kDa.