The nuclease gene of Vibrio vulnificus, vvn, is 696 bp long encoding a protein¡]Vvn¡^of 232 amino acids. Vvn is a periplasmic protein and is active in the oxidized form. DNA shuffling is a powerful method for in vitro mutational mechanism by homologous recombination with a low level of point mutation . DNA shuffling consists of four steps¡G¡]1¡^preparation of genes to be shuffled, ¡]2¡^random fragmentation with DNase I, ¡]3¡^fragment reassembly by primerless polymerase chain reaction¡]PCR¡^, and¡]4¡^amplification of reassembled products by a conventional PCR. The advantage of this process is that it can be used to rapidly evolve any protein, without any knowledge of its structure. The goal of this work was using DNA shuffling to generate a diversity of mutation in vvn within a short time. Followed by analyzing the DNase activity of periplasmic protein or in vivo, the mutants were divided into three groups for increase, decrease or no change in DNase activity. Randomly DNA sequencing vvn gene of fourteen transformed clones from the three groups showed only one clone has one base change with comparison to wild-type sequence. The mutation is at amino acid 22 of the N-terminus of Vvn, the change is from serine to isoleucine. The relative activity of mutant Vvn was 82 % in DNase and 59 % in RNase. The effect of a single amino acid change on the DNase and RNase activity of Vvn is different. It supports the postulation that there are two distinct but overlapping active sites exist in Vvn.

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is one of a group of bacteria known as cyanotrophs that exhibit the unique ability to grow on toxic cyanide as the sole nitrogen source. This ability has previously been genetically linked to a conserved cluster of seven genes (Nit1C), the signature gene (nitC) coding for a nitrilase enzyme. Nitrilases convert nitriles to ammonia and a carboxylic acid. Still, for the Pf11764 NitC enzyme (Nit11764), no in vivo substrate has been identified, and the basis of the enzyme's requirement for cyanide growth has remained unclear. Therefore, the gene was cloned and the enzyme was characterized with respect to its structure and function. These efforts resulted in the unique discovery that, aside from its nitrilase activity, Nit11764 exhibits nuclease activity towards both DNA and RNA. This ability is consistent with computer analysis of the protein providing evidence of a preponderance of amino acids with a high probability for RNA binding. A Nit11764 knock-out mutant was shown to exhibit a higher sensitivity to both cyanide (KCN) and mitomycin C, both known to induce chromosomal damage. Thus, the overall conclusion is that Nit11764, and likely the entire Nit1C gene cluster, functions as a possible repair mechanism for overcoming the damage inflicted on Pf11764 nucleic acids by toxic cyanide. Towards a further investigation of the Nit1C gene cluster in Pf11764, a second gene (nitH) annotated as a monooxygenase was also investigated. Interestingly, computer-based analysis shows that NitH also harbors a preponderance of RNA-binding amino acids. The nitH gene was cloned into an expression vector with the long-range goal of defining its role in CN utilization.

Indiana University-Purdue University Indianapolis (IUPUI) / Solid organ transplantation is severely limited by a shortage of available donor allografts. Pig-to-human xenotransplantation offers a potential solution to this growing problem. For xenotransplantation to achieve clinical relevance, both immunologic and physiologic barriers must be understood. Genetic modification of pigs has proven to be a valuable means of both studying and eliminating these barriers. The present body of work describes a method for greatly increasing the efficiency and precision of genome editing within the porcine genome. By combining non-integrating selection and homologous recombination of exogenous oligonucleotides, a method for rapidly creating genetic modification without reliance on phenotypic sorting was achieved. Furthermore this work employs the technique of CRISPR/Cas9-directed mutagenesis to create and analyze several new animal models of porcine-to-human xenotransplantation with respect to both immunologic and physiologic parameters. First, Isoglobotrihexosylceramide -a controversial glycan to the field of xenotransplantation- was studied in a knockout model and found not to affect human-anti-porcine humoral reactions. Second, a new combination of glycan modifications is described that significantly lowers the human anti-porcine humoral immune response. This model animal suggests that glycan-deletion alone will be sufficient to promote clinical application, and that conventional immunosuppression will be successful in mediating the human cellular response. Finally, two potential physiologic barriers to xenotransplantation are studied in genetically modified model animals. Xenogenic consumption of human platelets was studied across hepatic and renal organ systems; xenogenic platelet consumption was reduced by glycan modifications to the porcine liver while human platelet sequestration was not identified in the study of renal endothelium. Porcine FcRN –an essential receptor expressed in kidneys to maintain serum proteostasis- was studied as a final potential barrier to pig-to human renal transplantation. Because albumin is the primary driver of serum oncotic pressure, the protein-protein interaction of endogenous porcine FcRN and human albumin was studied. Porcine FcRN was found capable of binding human albumin under physiologic parameters. In summary, the results of the present work suggest that the salient barriers to clinical xenotransplantation have been removed and that porcine-to human renal transplantation may soon offer an answer to the current organ shortage.

Staphylococcus aureus encodes two extracellular nuclease enzymes, Nuc and Nuc2. Nuc is a secreted enzyme that is cut by signal peptidase (SpsB) at the cell membrane and is further processed into two active forms, NucA and NucB, by an unknown protease. Nuc2 is predicted to be a second extracellular nuclease based on sequence homology to the staphylococcal nuclease (SNase) and is tethered to the membrane with a N-terminal anchor. At the beginning of these studies, little was understood about the biological and physiological roles of Nuc and Nuc2 in S. aureus. The goal of this dissertation was to characterize the extracellular nuclease activity of S. aureus in order to better understand the contributions of Nuc and Nuc2 to the S. aureus life cycle. The studies presented in Chapter II focus on the role of Nuc in regulating S. aureus biofilm growth. The secreted forms of Nuc, called NucA and NucB, were first identified as anti-biofilm agents present in spent media from a S. aureus alternative sigma factor B (sigB) mutant. Regulation studies identified the major repressors and activators of nuc expression and showed that nuc is repressed under biofilm-forming conditions. By bypassing the native regulatory mechanisms using a nuc inducible plasmid, biofilm growth could be inhibited in a dose-dependent manner. Biofilm testing of nuc mutant strains across genetic backgrounds led to the observation that biofilm thickness increased two-fold in the absence of Nuc. More high molecular weight extracellular DNA (eDNA) accumulated in the nuc mutant compared to wild-type cells, indicating a direct link between Nuc and the availability of eDNA to contribute to the biofilm matrix. These studies showed that nuc expression is tightly regulated in S. aureus biofilms, and Nuc activity can greatly impact biofilm formation and maturation. In Chapter III, studies were performed to determine whether Nuc2 is an active nuclease in S. aureus and where the protein is localized in the cell. Upon initial comparison to Nuc, Nuc2 has 42% amino acid identity in the proposed SNase domain, and 7 of 9 residues known to be required for Nuc activity are conserved in Nuc2. Fluorescence microscopy of a Nuc2-sGFP translational fusion demonstrated the protein is localized to the cell membrane, and alkaline phosphatase fusion studies showed that the C-terminus of Nuc2 faces out of the cell. Fluorescence resonance energy transfer (FRET) assays facilitated the detection of low levels of Nuc2 activity on the S. aureus cell surface, demonstrating for the first time the enzyme is a functional nuclease, and mutations in the nuc2 gene eliminated this activity. Purification of recombinant Nuc2 also showed that enzyme has DNase activity that is calcium-dependent. Through the construction of Nuc/Nuc2 chimeric proteins, it was determined that localization to the cell membrane does not impair nuclease activity, and the low levels measured for Nuc2 on S. aureus cells is likely due instead to weak expression. The knowledge that Nuc2 is an active nuclease, localized to the cell surface, provides insight into the potential roles Nuc2 may play in a biofilm environment and during S. aureus infection.

DNA is the central storage molecule for genetic information in the cell. Therefore, the DNA must be protected from damage that will otherwise be passed on to future generations as deleterious mutations. Although many different pathways have evolved for repairing different classes of damage there are certain features that are common to all repair pathways. Generically, for DNA damage to be repaired it must first be recognized, then excised and replaced with undamaged DNA. DNA damage recognition is highly varied since specific interactions are required between the protein and the damaged DNA. DNA damage repair, paradoxically, requires the action of highly processive nucleases. The nucleases may digest hundreds if not thousands of nucleotides, sometimes for the repair of a single mutant nucleotide. We have chosen to focus on Exonuclease VII (ExoVII), one of the processive nucleases that have been implicated in the process of Mismatch Repair (MMR). ExoVII is a hetero-pentameric enzyme composed of one large subunit (XseA) and four small subunits (XseB). It has been previously characterized as a processive, single-strand specific nuclease able to digest DNA in either the 5'->3' or 3'->5' direction by a metalindependent mechanism. Early studies have shown that although ExoVII is a hydrolytic nuclease it was completely active in the presence of large amounts of EDTA and was strongly stimulated by phosphate. This feature is unusual because hydrolytic DNA nucleases typically function by a mechanism that requires coordination of a divalent cation. To further our understanding of the mechanism ExoVII we have identified and characterized the ExoVII homolog from Thermotoga maritima (T. maritima, Tm), a hyperthermophilic bacterium. The genes responsible for Tm ExoVII (TM1768 and TM1769) were cloned into an overexpression construct and the resulting proteins were overexpressed, co-purified and characterized. Consistent with previous studies, we found that Tm ExoVII is a processive, single-strand specific nuclease. Surprisingly, unlike Ec ExoVII, the T. maritima homolog was found to have an absolute requirement for the divalent cation magnesium and was strongly inhibited by the presence of either phosphate or sulfate in the reaction buffer. Using multiple sequence alignments of the large subunit we have identified a conserved core present within the C-terminal ExoVII_Large domain. This conserved core, RGGGx27GHx2Dx4Dx9P, although unique among nucleases, is reminiscent of a metal-coordinating hydrolytic active site. We have tested this putative active site using site-directed mutagenesis to create the TmD235A/TmD240A double mutant. This mutant protein was purified and the resulting protein was found to be inactive. We propose that this conserved core represents the metal-coordinating active site of all ExoVII homologs and that the group of E. coli-like homologs are unique in their EDTA resistance and anion (phosphate and sulfate) stimulation. Since ExoVII is a bi-directional nuclease (both 5'->3' and 3'->5' activity), and MMR is a bi-directional process, our model was that ExoVII was the primary nuclease associated with MMR. To test this model and determine if, in fact, a minimal conserved MMR pathway can be defined, we performed an analysis of the genomic occurrence profiles for the genes involved in MMR. To do this we have developed a bioinformatic application, Magma, which assists in the creation of a searchable relational database. Using Magma we have found that MutH, the enzyme responsible for generating a nick that directs MMR to excise the newly synthesized DNA strand including a DNA mispair, is only present in E. coli and a subset of gamma-proteobacteria, suggesting that MutH is not a core component of MMR. Instead, most organisms employ a nicking activity found in the MutL subunit. We also show that, although four nucleases have been implicated as having "redundant" roles in bacterial mismatch repair, RecJ is the primary nuclease responsible for degrading the mutated DNA strand and that 5'->3' single-strand exonuclease activity is a core MMR component. From this analysis, it appears that prokaryotic mismatch repair is more similar to eukaryotic mismatch repair than was previously thought, from the genetic and biochemical work done in E. coli. We offer a model for a universal minimal MMR system.

The periplasmic nuclease of Vibrio vulnificus, Vvn, has been purified to homogeneity by a one step purification procedure using chromatography on a SP Sepharose column. The purified enzyme showed different mobilities on reducing and non-reducing SDS-PAGE, suggesting that disulfide bonds are involved in the maintenance of a stable tertiary conformation of the protein. Vvn randomly cleaved single and double stranded DNA and RNA, and possessed endonucleolytic activity. The enzyme exhibited an optimal activity between pH 8.0 and pH 10.0, and the optimal temperatures for the DNase and RNase activity were 40 oC ¡V 60 oC and 40 oC ¡V 50 oC, respectively. The enzymatic activity was inhibited by EDTA and EGTA, indicating that Vvn was a metalloenzyme. The DNase and RNase activity of Vvn had different requirements for divalent cations. Chemical modification studies on Vvn revealed the involvement of lysine, arginine, tryptophan and carboxylate residues in the catalytic activity of the enzyme. The extents of inactivation of the DNase and RNase activity of Vvn by modification of the carboxylate group with EDC were different. Substrate DNA and RNA protected the DNase and RNase activity of Vvn from inactivation by PLP, PGO, NBS and EDC which modified lysine, arginine, tryptophan and the carboxylate group. Mg2+ could not protect the DNase and RNase activity of Vvn against the inactivation by PLP and PGO. Whereas Mg2+ protection was observed in NBS- and EDC-mediated inactivation of the DNase but not the RNase activity of Vvn . From these results, it is postulate that there may be two distinct but overlapping active sites, for the DNase and RNase activity, respectively.

Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities.

Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.

Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.

<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>