| Viruses that infect extreme hyperthermophilic archaea are unusual in their morphology, genome structure and sequence, and are model systems for understanding the biochemistry and molecular biology required for life at high temperatures. Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archeaon Sulfolobus islandicus at both extreme temperature (70°C--80°C) and acidity (pH 3). Despite ample data describing the SIRV2 genomic sequence, replication intermediates, transcription maps, and three-dimensional protein structures, it is unclear how SIRV2 and host S. islandicus proteins regulate and catalyze essential processes such as DNA replication and transcription.;Several approaches were taken to identify components of the SIRV2 replication initiation complex. Host DNA binding proteins that bind to SIRV2 hairpin DNA were identified by affinity purification. Several SIRV2 proteins (gp1, gp2, and gp31) interact with host replication machinery and may be involved in initiation. Further biochemical characterization of SIRV2 end processing demonstrated hairpin nicking activity in whole and fractionated SIRV2-infected S. islandicus cell extracts but components could not be further purified. A candidate initiation protein, SIRV2gp19, was characterized as a single-strand specific endonuclease which could initiate SIRV2 replication by nicking transient unpaired single-stranded hairpin regions, thus providing a 3'-OH for self-priming DNA synthesis.;Following nick initiation, leading strand DNA synthesis results in two linked SIRV2 genome copies separated by a cruciform structure. I have demonstrated that SIRV2 Holliday junction resolvase (Hjr) resolves dimer concatamers into single copies by cleaving cruciform DNA into linear products with nicked hairpin termini. SIRV2 Hjr was characterized biochemically and has a narrow substrate range and unique cleavage specificity in contrast to previously described cellular, bacteriophage, and viral Hjr. In addition, SIRV2 Hjr forms a complex with SIRV2 coat protein (gp26) and may nucleate viral particle assembly following genome resolution.;In summary, I have identified and characterized several putative components of SIRV2 replication initiation and genome resolution machinery and have proposed a unique model whereby the SIRV2 Holliday junction resolvase plays a dual role: resolving genome dimer concatamers and nucleating virion assembly. |
