Molecular, Biochemical, and Evolutionary Analysis of Single-Stranded Dna-Binding Proteins From the Mesophilic Archaeon Methanosarcina Acetivorans

190 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.One of the critical proteins involved in DNA replication across the three domains of life is single-stranded DNA-binding protein. At the replication fork, these proteins bind to stabilize and protect transiently exposed ssDNA from nucleases during DNA transactions. The ssDNA-binding protein is referred to as SSB in bacteria, whereas in eukaryotes and archaea it is known as replication protein A or RPA. Although the SSB's in the three domains differ in subunit composition, structural analyses have shown that SSBs and RPAs contain ssDNA-binding domains with a fold reminiscent of an oligonucleotide/oligosaccharide binding (OB) fold. In the Euryarchaeota, one of the subdomains in Archaea, RPAs display an unusual diversity. Single, multiple, and complex form RPAs have been described in Euryarchaeota. However, most euryarchaeotes contain a RPA composed of multiple OB folds and a zinc finger domain. Here, we have described a novel group of archaeal RPA that is composed of two OB folds and a zinc finger-like motif. The zinc finger-like motif is characterized by CX2CX8CX 2H, where x is any amino acid. We demonstrated that three invariant cysteines and a histidine in the zinc-finger of this novel RPA are essential for proper coordination of a zinc ion. Besides, the results suggested that the zinc finger module is important for the functional and structural integrity of the protein. Furthermore, we also demonstrated that the three distinct RPAs in M. acetivorans have different effects on DNA synthesis, strand exchange in homologous recombination, and flap endonuclease activity. This finding suggests that the Methanosarcinales might possess a "division of labor" among their three RPM in DNA transactions. In addition, we mimicked intramolecular and intermolecular homologous recombination process to engineer artificial RPA proteins through fusion of OB folds from M. acetivorans RPA I and M. thermoautotrophicus RPA. Functional studies of these engineered proteins suggested that domain fusion and module shuffling can lead to well folded polypeptides with most of the known properties of ssDNA-binding proteins. Based on these results we proposed evolutionary models to explain how intramolecular and intermolecular homologous recombination processes might have led to domain deletion and gene fusion to generate the diverse RPA homologs found in extant euryarchaeotes.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD