It is well known that the mitochondrial genome has a much higher spontaneous mutation rate than the nuclear genome. mtDNA mutations have been identified in association with many diseases and aging. mtDNA replication continues throughout the cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is required for replication and maintenance of the mitochondrial genome. However, it is not clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and nucleotide metabolism is associated with several human diseases. Clearly, to uncover the pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis, information is needed regarding dNTP biosynthesis and maintenance within mitochondria, and biochemical consequences of disordered mitochondrial dNTP metabolism. The studies described in this thesis provide important insight into these questions. First, we found that a distinctive form of ribonucleotide reductase is associated with mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP synthesis within mitochondria. Second, we found that long term thymidine treatment could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from thymidine treatment could account for the spectrum of mtDNA point mutations found in Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results support the proposed pathogenesis of this disease. Third, we found that normal intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity studies show that these imbalanced pools can stimulate base substitution and frameshift mutations, with a substitution pattern that correlates with mitochondrial substitution mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases. Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by insufficient transport of dNTPs into mitochondria resulting from a loss-of-function mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous mutant cells extracted from Dnc gene knockout mouse embryos. These results do not support the proposed pathogenesis of this disease and indicate that the DNC protein does not play a crucial role in the maintenance of intramitochondrial dNTP pools. / Graduation date: 2005

Graduation date: 1999

A sophisticated computer program is developed to analyze flow linear dichroism data on nucleic acids for individual base inclinations. Measured absorption and linear dichroism data for synthetic AT and GC polymers and natural DNAs are analyzed. The reliability of the program is tested on data for the synthetic polymers, and the results are similar to earlier, more straightforward analyses. For the first time, specific base inclinations are derived for all bases individually from the linear dichroism data for natural deoxyribonucleic acids. For B-form DNA in aqueous solution at moderate salt concentrations, the inclinations from perpendicular are as follows: d(A)=16.1 �� 0.5; d(T)=25.0 �� 0.9; d(G)=18.0 �� 0.6; d(C)=25.1 �� 0.8 deg. Our results indicate that the bases in synthetic and natural DNAs are not perpendicular to the helix axis, even in the B form. The mathematical bases and numerical analyses are presented in detail since both are the keys for successful spectral decompositions in this study, and could be applied to nonlinear optimization problems encountered in other types of biochemistry and biophysics measurements. The interplay between computer programming and scientific measurements can not be overemphasized for modern research. / Graduation date: 1994

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

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.

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

A crucial factor in determining the accuracy of DNA replication is maintenance of a balanced supply of deoxyribonucleoside triphosphates (dNTPs) at replication forks. Perturbation of dNTP biosynthesis can induce dNTP pool imbalance with deleterious genetic consequences, including increased mutagenesis, recombination, chromosomal abnormalities and cell death. Using the T4 bacteriophage system, I investigated the molecular basis of mutations induced by imbalanced dNTP pools in vivo. Two approaches were adopted to disturb dNTP biosynthesis: 1) using mutations which affect the deoxyribonucleotide biosynthesis pathway; 2) exogenously supplying mutagenic deoxyribonucleoside analogs which are then taken up by cells and are metabolized to dNTPs. The levels of dNTPs under different conditions were measured in crude extracts of phage-infected cells, while mutagenic effects were quantitated by analysis of certain rII mutations, thought to revert to wild type along either GC-to-AT or AT-to-GC transition pathways. The mutation pathways stimulated by dNTP pool perturbations were confirmed by direct DNA sequencing after amplification of template by the polymerase chain reaction (PCR). By replacing phage ribonucleotide (rNDP) reductase with the host, Escherichia coli, rNDP reductase, in phage-infected cells, I examined the mechanism of mutation induced by the thymidine analog 5- bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to- AT transition mutations were stimulated many hundred-fold when cells were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions were stimulated predominantly when T4 reductase was active, while ATto- GC transitions were stimulated more when E. coli reductase was active. By examining the control by dNTPs on CDP reduction, I found that the T4 rNDP reductase is substantially inhibited by either BrdUTP or dTTP in crude enzyme extracts. These experimental results are consistent with the hypothesis that mutagenic effects of BrdUrd are based on dNTP perturbations, supporting the model that rNDP reductase is a major determinant of BrdUrd mutagenesis. I also studied the mutator phenotype of one temperature-sensitive conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4 deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions induced by ts LB3 at a semipermissive temperature (34° C). Sequence analysis of revertants from the most sensitive gene marker, rII SN103, showed that either cytosine within the mutated triplet can undergo change to either thymidine or adenine, supporting a model in which mutagenesis induced by ts LB3 at a semipermissive temperature is based on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine triphosphate (hm-dCTP) caused by the temperature-labile dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations. However, no significant dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected cells even when cells were grown at the semi-permissive temperature, suggesting that imbalanced dNTP pools occurred only locally, close to replication forks. These results support a model of dNTP "functional compartmentation", in which DNA replication is fed by a small and rapidly depleted pool, with the bulk of measurable dNTP in a cell representing a replication-inactive pool. To further characterize the mutagenic specificity and DNA site specificity induced by T4 ts LB3, I developed a fast forward mutation approach using thymidine kinase as a marker gene. The studies confirmed that the principal mutagenic effect induced by ts LB3 is C-to- T transition, while C-to-A transversion mutagenesis also occurs. Analysis of DNA sequences around each mutation also suggests that local DNA context influences mutation frequency. / Graduation date: 1991

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

Recently, DNA computing is one of powerful tools that can be designed for solving NP-complete problems. The powers of DNA computing are that it has great ability of massive data storage and it can process those data in parallel. Some of hard problems, such as the traveling salesperson problem and the Hamiltonian cycle problem, have been solved with the brute force method in DNA computing. After DNA computing is performed, all feasible solutions for the problem are stored implicitly in the tubes. However, the correct answer still cannot be extracted or reported absolutely, because that the concentration of the correct solutions might be lower than other bad solutions. In this paper, we will increase the concentration of the correct answer for fault-tolerant ability.

Homologous genetic recombination is a central feature of meiosis in most sexually dividing organism. It leads to the establishment of new linkage relationships between genes and is crucial for the successful completion of meiosis. It is also necessary for a variety of important cellular events like immunoglobulin rearrangement, repair of chromosome damage, gene amplification, gene expression and sister chromatid exchange. Knowledge of the mechanisms of homologous recombination has come from extensive genetic studies on fungi. These studies have led to the formulation of various models which explain the genetic observations. Analysis of the fate of transfected DNA molecules into cells have revealed the existence of additional recombinational pathways in eukaryotes. The biochemistry of homologous recombination has mainly focused on the isolation and characterisation of the recombinase activity from various systems. The E. cold RecA protein remains the best characterised recombinase or pairing protein till date and its reactions in vitro, in particular the strand-transfer reaction, have laid the framework for isolating such proteins from eukaryotic systems. However, limited success has been achieved in the purification and characterisation of pairing activities from mammalian cells. The objective of the present study was to isolate and characterise a pairing activity from, a meiotic tissue of a mammalian system - the rat testis. Meiosis constitutes one of the important phases of the process of spermatogenesis. In rat, the various stages of spermatogenesis (the meiotic stages and spermiogenesis) can be broadly demarcated by the age of the rat. The pachytene stage of meiosis in rat is spread over nine days. Rat testicular nuclear extracts from animals, aged 38-42 days (the stages of spermiogenesis were absent and a significant proportion of the cells were in the pachytene stage during this period), were fractionated sequentially on phosphocellulose, heparin-agarose and ssDNA-cellulose columns. Fractions were tested for the ssDNA-dependent formation of slow-moving products on an agarose gel, using the strand-transfer away with M13mp19 RF III and ssDNA substrates. A partially purified fraction, which catalysed the formation of such products, was obtained from the ssDNA-cellulose column on eluting with 0.6 M KCI. In order to characterise the products formed by this partially purified fraction, they were analysed by electron microscopy. The majority of the observed structures represented multimers of the linear substrate. A significant proportion of the products resembled forked (Y-branched) structures and paired (duplex-duplex paired and 6s-duplex paired) structures. Length measurement of the multimers indicated the presence of a ligase activity in the partially purified fraction. The arms of the Y-branched structures were equal in length. The length of the molecule from the end of the stem to the end of either of the arms was the same as that of a monomer. The Y-branched structures were duplex in nature along their entire length. Exonucleases were not detected in the fraction; hence, these structures might actually represent paired structures. Typical r- and &-structures were not observed. The EM studies indicated that the slow-moving products, observed on the gel, might be ligated structures. The mobility of these products were found to be similar to those formed by a standard ligase - the T4 DNA ligase. Restriction digestion of the junctions of the ligated products revealed that the ends were intact and there was no nucleotide loss, as seen in the case of ligases involved in nonhomologous recombination. In order to characterise the type of ligase present, radiolabelled a-ATP was used to form the ligase-AMP adduct which was then separated by SDS-PAGE. Autoradiography revealed the presence of a major 100 kDa polypeptide (most probably DNA ligase I or 111), with a minor 65 kDa polypeptide (most probably DNA ligase 11). The latter, in turn, was found to be enriched in the ssDNA-cellulose fraction eluting with 0.8 M KC1. The formation of the ligated products was strictly dependent on the presence of ssDNA. Other characteristics of the ligase activity included the substitution of ATP by UTP. The ligase present in the 0.8 M fraction was found to be inhibited by ssDNA. Experiments to test the heat - stability of the products revealed the presence of a ssDNA-aggregating activity in the partially purified fraction. This aggregation was found to be specific for linear ssDNA. In the strand-transfer assay, the first step involves the transfer of the ssDNA circle on the linear duplex substrate accompanied by the partial displacement of the non-complementary strand of the duplex. It was reasoned that the linear end generated (partially-displaced strand) would be aggregated by the linear ssDNA-aggregating activity, thereby preventing the progression of the reaction and accounting for the absence of V - and a-structures under the EM. The formation of such aggregates was therefore monitored on the gel, using radiolabelled linear dsDNA. The formation of theme aggregates was found to be dependent on ssDNA and homology. ATP was essential for the reaction, though ATP hydrolysis was not necessary, since ATPW could substitute for ATP in the reaction. This suggested the presence of a pairing activity in the partially purified fraction. The aggregating activity was found to be enriched in the 0.5M KCl-fraction from the ssDNA-cellulose column. The molecular weight of the enriched polypeptide was found to be similar to that of histone Hl. Western blot analysis of the polypeptide with monospecific antibodies against histone Hl a and a comparison of the protein fingerprinting pattern of the polypeptide with that of histone Hla revealed the identity of the polypeptide to be histone Hla. Formation of stable protein-free aggregates of linear ssDNA was found to be a common property of histone H1 subtypes. This aggregation of linear ssDNA (non-complementary) did' not require ATP or Mg2+. Purified hietone H1 subtypes, however, could not cause homology-dependent aggregation of the linear duplex substrate. This study thus indicates the presence of a pairing activity and a ligase activity in a partially purified fraction from rat testicular nuclear extracts. Unlike other eukaryotic pairing activities characterised so far, this preparation is free from exonucleases. The homology-dependent duplex DNA aggregates, observed in this study, may therefore represent intermediates actually generated by the pairing activity.