Forensic genotyping uses a multiplex short tandem repeat (STR) assay to amplify deoxyribonucleic acid (DNA) samples. One of the artifacts mostly commonly encountered in forensic DNA analysis is stutter, which are non-specific products from the polymerase chain reaction (PCR) that are typically one repeat unit shorter in length than the allelic amplicon. While stutter peaks are typically no taller than 10% of the parent peak on electropherograms, their peak heights can fall into similar ranges as minor contributor alleles in mixtures, creating a problem of how to distinguish artifacts from true allele peaks in these situations. One way to potentially address this issue is to find a PCR method that produces a much lower amount of stutter than the method currently used, which involves amplifying samples with commercial PCR kits designed for forensic applications. These kits all use some form of Taq DNA polymerase (derived from Thermus aquaticus). In an effort to examine whether the type of enzyme used in an assay affects the resulting stutter rates observed, the existing GlobalFiler™ PCR Amplification Kit (Applied Biosystems) protocol for forensic multiplex STR assays was modified to test different types of enzymes. This was done by amplifying the same DNA sample with GlobalFiler primers and different commercial proofreading enzymes and their accompanying reaction buffer using manufacturer-recommended PCR parameters. The DNA sample originated from a buccal swab that was extracted on the EZ1® Advanced (Qiagen). The DNA solution was quantified using the Quantifiler™ Duo DNA Quantification Kit (Applied Biosystems) on the 7500 Real-Time PCR System (Applied Biosystems). In order to first establish the validity of switching out enzymes in an established protocol, a DNA sample was amplified with the Type-it® Microsatellite Kit (Qiagen), another Taq-based kit that is also marketed for use in multiplex STR assays. After a complete profile was successfully generated, research proceeded with testing various high-fidelity DNA polymerases. Some of the enzymes tested were known to be Pyrococcus-like while others were fused to a DNA-binding domain to enhance processivity. Taq polymerases tend to produce products with 3’adenine-overhangs while proofreading enzymes produce blunt-ends. This change caused a one base pair difference in the resulting amplicon lengths, which was accommodated by manually assigning genotypes after results from fragment analysis by capillary electrophoresis using a 3130 Genetic Analyzer (Applied Biosystems) were interpreted by the GeneMapper™ software (Applied Biosystems). Additional amplification kits tested were: the UCP HiFidelity PCR Kit (Qiagen), Phusion™ Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific), Platinum™ SuperFi™ II DNA Polymerase (Invitrogen), iProof™ High-Fidelity DNA Polymerase (Bio-Rad), Q5® High-Fidelity DNA Polymerase (New England Biolabs), and TruFi™ DNA Polymerase (Azura Genomics). Most of the kits produced profiles exhibiting a high degree of uneven amplification and varying levels of allelic dropout. In addition, all of the kits tested had much shorter peak heights compared to using GlobalFiler. Changing the type of enzyme used in an established protocol was found to be less straightforward than anticipated. Due to the poor quality results obtained in the first pass of trials, a few kits were selected to undergo optimization in the hopes of achieving higher quality results from which further analyses, such as comparing stutter rates, could be more reliably conducted. Both altered reagent amounts (higher enzyme concentration, higher DNA input mass) and different PCR parameters (decreased denaturation temperature, varying annealing temperature, decreased extension temperature, longer extension cycles, and longer final extension stage) were assessed. Only an increase in extension cycling time was found to produce better peak heights while maintaining balanced amplification of most of the targeted loci. Initial samples amplified with the Phusion enzyme exhibited multiple non-specific artifacts that were not stutter. Raising the annealing temperature for that enzyme’s protocol eliminated this issue. Therefore, higher annealing temperatures were pre-emptively used for several of the other enzymes tested. One of the explanations proposed for the uneven amplification observed is the presence of inhibitors in the commercial buffers used affecting downstream capillary electrophoresis. The Q5 High-Fidelity and TruFi DNA polymerases produced the best quality profiles; the UCP HiFidelity PCR Kit had the poorest results. Preliminary results indicated that none of the protocol alterations implemented significantly decreased stutter rates, nor was any one commercial enzyme found to have consistently lower stutter rates than the GlobalFiler kit. Due to the low number of trials carried out, the findings from this study require more replications with a wider variety of DNA polymerases to confirm that the type of enzyme used in an assay does not affect stutter rates.

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase. Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase λ (pol λ), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol λ can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol λ, we also show that the unique proline-rich region in pol λ plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol λ interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol λ. Taken together, these data strongly support that pol λ functions in DNA polymerization events during NHEJ.

The work in the Brown laboratory has two long-range objectives. Both are derived from an interest in the replication of the genome of Gram-positive eubacteria. One objective is to gain a deeper understanding of the structure and function of DNA polymerase III, the unique species of DNA polymerase which is essential for chromosome replication. The second objective, the one from which this thesis is derived, is to determine whether a selective inhibitor of this DNA polymerase can serve as a basis for producing a new generation of clinically useful Gram-positive-selective antimicrobial agents. The polymerase III-specific inhibitor prototypes investigated in this work are members of a family of simple 6-substituted uracils. The following members of this family, TMAU and EMAU, were used as platforms for the manipulation of the N3 atom (arrow), the only ring component which could be substituted without severe reduction of inhibitory activity. The N3 position was substituted with a series of alkyl groups of increasing size. The resulting structure-activity relationships at the level of the polymerase was consistent with the presence of an N3-specific subdomain within the inhibitor binding site which could accommodate a wide variety of substituents. Although specific alkyl substituents at N3 also significantly enhanced the antibacterial potency of TMAU and EMAU, the respective compounds were found to have insufficient aqueous solubility for successful application in in vivo infection. To increase aqueous solubility, the N3 atom of the EMAU platform was substituted with selected hydroxy- and methoxyalkyl groups. The latter agents retained both anti-polymerase and antibacterial activity, and, as expected, they displayed a combination of lipid and aqueous solubility favorable to efficacy in in vivo infection. Two of the agents, N3-hydroxypropyl- and N3-methoxypropyl-EMAU were examined for their ability to protect mice from lethal staphylococcal infection. Both were found to be active in this model. In sum, the results of this work demonstrated, for the first time, that: (1) the eubacterial replication-specific DNA polymerase III is a valid target for antibiotic development, and (2) the N3-substituted 6-anilinouracil platform has strong potential as a basis for novel antibiotics useful against Gram-positive bacterial infection.

The major research interest of our laboratory is focused on the replication-specific DNA polymerase III (pol III) family in Gram+ bacteria, and has used Bacillus subtilis (BS) as the primary model enzyme for study. The long range objective of the work of the laboratory is to gain a deeper understanding of the structure and function of Gram+ bacterial DNA polymerase IIIs, a structurally unique class of DNA-dependent DNA polymerase which are uniquely susceptible to inhibition by a specific class of dGTP analogs. The project described in this thesis dissertation deals specifically with the pol III of the Gram+ organism Staphylococcus aureus, and involves the isolation and characterization of DNA pol III from this clinically relevant pathogenic bacterium. A homology-based strategy was devised to clone the structural gene specifying DNA polymerase III of Staphylococcus aureus, SA polC. SA polC was found to contain a 4305-bp open reading frame (ORF) encoding a 162.4 kDa polypeptide, and mapped between Ω1074[Tn551] and recA/ngr on the genome map of S. aureus NCTC 8325. The 1435 codon ORF was engineered into the E. coli expression plasmid pBS(KS) under the control of the lac promoter and its repressor. The translational signals of SA polC were reengineered using expression cassette PCR (ECPCR) to optimize the in vitro expression of SA polC in E. coli. Derepression of E. coli transformants carrying the recombinant vector generated high level expression of active recombinant pol III. The recombinant SA pol III was purified to greater than 98% homogeneity and was shown by N-terminal amino acid analysis to be the bona fide product of the 4305-bp SA polC ORF. The physical and catalytic properties of recombinant SA pol III and its responsiveness to inhibitors of the HPUra type were similar to those of Bacillus subtilis (BS) pol III. Comparative structural analysis of the primary structure of SA pol III and the pol IIIs of B. subtilis and the Gram+ relative Mycoplasma pulmonis indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of E. coli pol III and these three Gram+ enzymes suggested a specific evolutionary relationship between the pol IIIs of Gram+ and Gram- bacteria.

Plants have two organelles outside the nucleus which carry their own DNA, mitochondria and chloroplasts. These organelles are descendants of bacteria that were engulfed by their host according to the endosymbiotic theory. Over time, DNA has been exchanged between these organelles and the nucleus. Two polymerases, DNA Polymerases Gamma I and II, are encoded in the nucleus and remain under nuclear control, but are transported into the mitochondria and chloroplasts. DNA polymerases gamma I and II are two organelle polymerases which have been studied through sequence analysis and shown to localize to both mitochondria and chloroplasts. Little has been done to characterize the activities of these polymerases. Work in tobacco showed the homology of these polymerases to each other and to DNA Polymerase I in bacteria. They have been characterized as being part of the DNA Polymerase A family of polymerases. In my research I have studied the effect of T-DNA insertions within the DNA Polymerase Gamma I and II genes. Since these DNA Polymerases are targeted to the mitochondria and chloroplasts, I studied the effect of knocking out these genes. A plant heterozygous for an insert in DNA Polymerase Gamma I grows slightly slower than wild type plants with an approximately 20% reduction in mitochondrial and chloroplast DNA copy number. A plant homozygous for an insert in this same gene has a drastic phenotype with stunted plants that grow to around 1 inch tall, with floral stems, and have an approximately 50-55% reduction in mitochondrial and chloroplast DNA copy number. Wild type plants can grow to a height of 12-18 inches with floral stems as a comparison. A plant heterozygous for an insert in the DNA Polymerase Gamma II gene grows slightly slower than wild type plants and has an approximately 15% reduction in mitochondrial DNA copy number and a 50% reduction in chloroplast DNA copy number. These plants also produce much less seed than do other mutants and wild type plants.

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