<p>DNA partition or segregation is the process that ensures the stable inheritance of genomic material. The majority of the bacterial plasmid and some chromosomal partition systems utilize ParA Walker-box-based partition systems. These systems require three components: a DNA centromere site, the ParA ATPase, and the ParB centromere binding protein. ParB binds to the centromere to form the partition complex, which then recruits the motor protein ParA. ParA mediates the partition of replicated DNA by a still poorly understood mechanism. Notably, recent data indicates that ParA Walker-box-based partition systems are employed not only by bacterial plasmids and chromosomes but also DNA elements in archaea. The work in this thesis focused on a homolog of the ParA protein from the first identified archaeal plasmid partition system, located on the plasmid pNOB8. pNOB8 plasmid is harbored in the thermophilic archaeaon, Sulfolobus solfataricus. The goals of this work were to structurally and biochemically characterize the ParA homolog to gain insights into its function.</p><p>Towards these goals, the structure of the ParA homolog was solved by X-ray crystallography in its apo and ADP bound states to resolutions of 2.45 Å and 2.73 Å, respectively. The overall structure was similar to bacterial ParA proteins. We next demonstrated that, similar to bacterial ParA proteins, this ParA homolog harbored ATP-dependent nonspecific DNA capabilities by using fluorescence polarization based DNA binding assays. By mutating the residues in the deviant Walker A motif, we were able to demonstrate the importance of ATP binding in its DNA binding function. Moreover, characterization of ATP and ADP binding were performed using ITC. Finally, we observed that ParA was able to form polymers in the presence of ATP, using negative stain electron microscopy. Our findings provide evidence that ParA Walker-box-based partition systems, which are the most common systems in bacteria, appear to also be found in archaea.</p> / Dissertation

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Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 13, 2010). Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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(S)-3-O-Geranylgeranylglyceryl Phosphate Synthase (GGGPS) is a TIM (βα)8 barrel protein found in Archaea and the enzyme catalyzing the first step in the biosynthesis of archaeal membrane lipids. The TIM (βα)8 barrel protein fold is thought to have evolved by duplication and fusion of (βα)4 half barrels. We propose that the GGGPS has also evolved from (βα)4 half barrels. One way to test this hypothesis is to generate putative half-barrels experimentally. GGGPS from Archaeaglobus fulgidus, is a dimer of (βα)8 barrels. Thus, before constructing half barrels, a stable monomer is needed to be engineered. Introducing three substitutions into the dimer interface formed the GGGPS monomer. AUC showed ~50 % of the protein is in the monomeric state. CD experiments confirmed that the engineered protein was properly folded but had decreased thermal stability. In an enzymatic assay, the monomeric GGGPS protein proved as active as the WT protein on a subunit basis.

(S)-3-O-Geranylgeranylglyceryl Phosphate Synthase (GGGPS) is a TIM (βα)8 barrel protein found in Archaea and the enzyme catalyzing the first step in the biosynthesis of archaeal membrane lipids. The TIM (βα)8 barrel protein fold is thought to have evolved by duplication and fusion of (βα)4 half barrels. We propose that the GGGPS has also evolved from (βα)4 half barrels. One way to test this hypothesis is to generate putative half-barrels experimentally. GGGPS from Archaeaglobus fulgidus, is a dimer of (βα)8 barrels. Thus, before constructing half barrels, a stable monomer is needed to be engineered. Introducing three substitutions into the dimer interface formed the GGGPS monomer. AUC showed ~50 % of the protein is in the monomeric state. CD experiments confirmed that the engineered protein was properly folded but had decreased thermal stability. In an enzymatic assay, the monomeric GGGPS protein proved as active as the WT protein on a subunit basis.

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

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