Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

The retinoblastoma tumor suppressor protein (RB) is an important regulator of the cell cycle and development. Significantly, RB is inactivated in a majority of human cancers. Thus, elucidating the function of RB will give us a better understanding of how it prevents cancer. Many decades of research have yielded a detailed understanding of the role of RB in cell proliferation through transcriptional repression of target genes. However, the precise mechanisms of its action in many cellular pathways are poorly understood, including the control of DNA replication and post-transcriptional control of gene expression. Drosophila melanogaster presents a simplified genetic system to study cancer genes. Several published observations have suggested a role for RB in regulating DNA replication. Interestingly, other data indicate that RB associates with RNA processing factors. I have characterized novel protein-protein interactions with the Drosophila retinoblastoma tumor suppressor homologue Rbf, with an emphasis on its poorly characterized N-terminal domain. I describe the interaction of Rbf with the origin recognition complex, indicating a unique connection to DNA replication control. I also show that Rbf interacts with the RNA binding protein Squid, and review the literature that suggests potential role of RB/E2F in the control of RNA processing. The ability to control RNA processing may be an additional, unappreciated mode of gene regulation by RB. A focused study of the uncharacterized amino-terminal domain of Rbf has revealed new details about the retinoblastoma tumor suppressor in cell regulation, including DNA replication and RNA processing.