Friction Bit Joining (FBJ) is a new technology used primarily in joining dissimilar metals. Its primary use has been focused in the automotive industry to provide an alternative joining process to welding. As automotive manufacturing has continually pushed toward using dissimilar materials, new joining processes have been needed to replace traditional welding practices that do not perform well when materials are not weld compatible. FBJ meets these needs perfectly as it provides strength as well as the ability to join materials of almost any kind.The purpose of this research was to explore different applications of the FBJ process. Traditionally FBJ has used a steel bit to drill through a thin piece of aluminum and weld to a piece of steel behind the aluminum. This research explored a different application of FBJ by using a steel bit to drill through multiple pieces of aluminum and weld to a small steel bit on the backside of the aluminum. The primary goal of this research was to answer two questions. (1) How does drilling impact peak weld strength and (2) Does an optimal shank diameter exist in terms of peak weld strength? As in other research, no universal parameters were found for optimization of lap shear, cross tension and t-peel tests. Drilling was found to be an important factor in peak weld strength. Number of flutes on the consumable steel bit was varied to see the impact of better and worse chip clearance ability. Increasing number of flutes was found to positively impact peak weld strength to a point. Optimal number of flutes was found to be different for each type of testing. It was found that there was an optimal bit head to bit shank diameter ratio that optimized peak weld strength. Again the optimal diameter was different for each test. Bits of different diameters were created and then tested to measure the impact of varying shank diameters on peak weld strength. It was found that there was a strength tradeoff between two localized joint areas in diameter testing. Decreasing the shank diameter increased the amount of overlap formed by the bit head over the top coupon. This shifted strength to the bit head region. While this strengthened the bit head region of the joint, strength was sacrificed in the bit-nut intersection. This tradeoff was consistently found in all test types.

DNA replication is one of the most fundamental processes in all living organisms. Its semi-discontinuous nature dictates that the lagging strand is synthesized in short fragments called Okazaki fragments. In eukaryotes, each Okazaki fragment is initiated by an ~ 30-40 nucleotide-long RNA-DNA hybrid primer that is synthesized by Pol α-primase complex. To ensure genomic stability, the RNA primer has to be excised, any misincorporations by Pol α have to be corrected for and finally the resulting nick has to be sealed generating a contiguous strand. This feat is accomplished by a highly coordinated and regulated process called Okazaki fragment maturation. At the center of this process are 5’ nucleases, which are structure-specific nucleases that catalyze the incision of phosphodiester bonds one nucleotide into the 5’ end of ssDNA/dsDNA junctions. Previous structural and biochemical studies have shed some light on the mechanism of FEN1 substrate recognition, its catalysis and regulation. However, many gaps in our understanding of this remarkable nuclease still persist. Moreover, the choice between the short- and long-flap pathways is still elusive. Finally, the mechanism of the coordination among the different enzymatic activities of the polymerase, the nuclease and the ligase during Okazaki fragment maturation is still debatable. In this work, we set out to study FEN1 substrate recognition, catalysis and regulation using single molecule techniques. We show that FEN1 employs a sophisticated substrate recognition mechanism through which it actively distorts the DNA to ~100˚ bent angle. It also displays a remarkable selectivity towards its cognate substrate and avoids off-target substrate by a lock-down mechanism that commits the enzyme for catalysis on cognate substrates while promoting the dissociation of non-cognate substrates. We further characterized FEN1 reaction from substrate binding/bending to product handoff and built a comprehensive kinetic scheme that shows FEN1 releasing its product in two steps. Finally, we uncovered an unprecedented role of FEN1 in the choice between short- and long-flap pathways.

Friction Bit Joining (FBJ) is a new technology used primarily in joining dissimilar metals. Its primary use has been focused in the automotive industry to provide an alternative joining process to welding. As automotive manufacturing has continually pushed toward using dissimilar materials, new joining processes have been needed to replace traditional welding practices that do not perform well when materials are not weld compatible. FBJ meets these needs perfectly as it provides strength as well as the ability to join materials of almost any kind.The purpose of this research was to explore different applications of the FBJ process. Traditionally FBJ has used a steel bit to drill through a thin piece of aluminum and weld to a piece of steel behind the aluminum. This research explored a different application of FBJ by using a steel bit to drill through multiple pieces of aluminum and weld to a small steel bit on the backside of the aluminum. The primary goal of this research was to answer two questions. (1) How does drilling impact peak weld strength and (2) Does an optimal shank diameter exist in terms of peak weld strength? As in other research, no universal parameters were found for optimization of lap shear, cross tension and t-peel tests. Drilling was found to be an important factor in peak weld strength. Number of flutes on the consumable steel bit was varied to see the impact of better and worse chip clearance ability. Increasing number of flutes was found to positively impact peak weld strength to a point. Optimal number of flutes was found to be different for each type of testing. It was found that there was an optimal bit head to bit shank diameter ratio that optimized peak weld strength. Again the optimal diameter was different for each test. Bits of different diameters were created and then tested to measure the impact of varying shank diameters on peak weld strength. It was found that there was a strength tradeoff between two localized joint areas in diameter testing. Decreasing the shank diameter increased the amount of overlap formed by the bit head over the top coupon. This shifted strength to the bit head region. While this strengthened the bit head region of the joint, strength was sacrificed in the bit-nut intersection. This tradeoff was consistently found in all test types.

Fluorescence-based assays have gained an ever-increasing popularity in life sciences. One of these rapidly emerging techniques is Protein Induced Fluorescence Enhancement (PIFE). Traditional explanations of PIFE focused exclusively on the role of the protein and largely neglected the role of the mediator DNA. In the same time, the existing models of PIFE were denying its exactly opposite effect. In the first part of the current dissertation we focus on a better understanding of PIFE, stimulated by the direct observation of its opposite effect, Induced Fluorescence Enhancement Quenching (PIFQ). This study offered us the leverage for obtaining on-demand fluorescence modulation in cyanine dyes. The following two chapters harvest this control over fluorescence modulation to generate two biotechnology applications: a sensitive potassium sensor with embedded fluorescent transducer, and a simple protocol for the fluorescent detection of His-tagged proteins. In the last part, a variety of fluorescence tools including Förster resonance energy transfer, fluorescence enhancement, and fluorescence quenching are employed for a much more complex task; to demystify the behavior of the human Maturation of Okazaki Fragments (MOF) machinery. First, we reconstituted the human MOF reaction and showed that it behaves considerably different than its well-established yeast homolog. Subsequently, our toolbox of fluorescence-based assays was used to pinpoint the kinetics and dynamics that lead to this unexpected MOF behavior.

Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol a), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase a tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol a tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) <i>Saccharomyces cerevisiae</i> helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

DNA replication is essential for the propagation of all living organisms. The ability of a cell to accurately duplicate its entire genome is dependent upon the activity of numerous proteins. Identifying the molecular mechanisms by which these proteins act, and determining how they are physically and functionally coordinated at sites of active DNA replication, is central to understanding this essential cellular process. Archaea possess a DNA replication machinery which is ancestral to the one present in eukaryotes, and thus these organisms serve as simplified model systems for understanding the complexities of eukaryotic DNA replication. This thesis investigates the molecular mechanisms underlying Okazaki fragment maturation in the crenarchaeon Sulfolobus solfataricus, which is essential to the completion of lagging strand DNA replication. Reconstitution of Okazaki fragment maturation in vitro demonstrated that the activities of three enzymes – PolB1, Fen1, and Lig1 – are required for this process in S. solfataricus. Furthermore, it was shown that optimum coordination of their three distinct activities is dependent on the ability of PolB1, Fen1 and Lig1 to simultaneously interact with a single PCNA ring, providing evidence for a mechanism of multi-enzyme coordination which may be universally employed by DNA sliding clamp proteins. The importance of protein flexibility in the accommodation of multiple proteins around a single PCNA was also investigated. Finally, the physical coordination of one of these key maturation enzymes – PolB1 – with other replisome proteins was examined. It was demonstrated that PolB1 exists in a trimeric complex in vivo with two previously unidentified factors, raising the possibility of uncharacterised activities and interactions for this crucial enzyme. Taken together, these data provide new insights into functionally important protein-protein interactions within the archaeal replisome, and facilitate a greater understanding of the DNA replication machinery in both archaea and eukaryotes.

Previous studies suggest a degree of homology between eukaryotic replication, transcription and translation proteins and archaeal ones. Hence, Archaea are considered a simplified model for understanding the complex molecular machinery involved in eukaryotic DNA metabolism. DNA replication in eukaryotic cells is widely studied. In recent years, DNA replication studies expanded on the archaeal DNA replication machinery. P. abyssi was the first archaeon whose genome was fully sequenced. Genome sequencing and comparative genomics have highlighted an MCM-like protein in P. abyssi. In this study, I report the biochemical and structural characterisation of PabMCM. PabMCM is explored as model for understanding more complex eukaryotic MCM proteins and unravelling the biochemical mechanism by which MCM proteins release their helicase activity. The crenarchaeon Sulfolobus solfataricus possesses a simplified toolset for DNA replication compared to Eukaryotes. In particular, S. solfataricus has a subset of the eukaryotic Okazaki fragment maturation factors, among which there are a heterotrimeric DNA sliding clamp, (the proliferating cell nuclear antigen, PCNA), the DNA polymerase B1 (PolB1), the flap endonuclease (Fen1) and the ATP-dependent DNA ligase I (LigI). PCNA functions as a scaffold with each subunit having a specific binding affinity for each of the factors involved in Okazaki fragment maturation. Here, the 3D reconstruction of PCNA in complex with the Okazaki fragment maturation proteins PolB1, LigI and Fen1 is reported.

The rate of DNA mutation is known to fluctuate across the genome but the patterns of mutation rate variation and molecular causes are poorly defined. It is important to understand these patterns of mutation as they influence where deleterious mutations are likely to arise and how rapidly sequences are likely to accumulate change between species, a measure often used as a proxy for functional constraint. In this work I investigate the relationship between DNA replication and apparent mutation hotspots adjacent to transcription factor binding sites. In eukaryotes both DNA strands are replicated simultaneously, the leading strand as a continuous stretch and the lagging strand as a series of discrete Okazaki fragments that are subsequently ligated together. Some transcription factors are able to bind the DNA lagging strand during replication and act as a partial barrier to DNA polymerase, resulting in the accumulation of Okazaki fragment junctions adjacent to these sites. I find that mutation rate is correlated genome wide with Okazaki junction frequency, suggesting that Okazaki junction processing may be error-prone. We present a mechanistic hypothesis to explain this locally elevated mutation rate and propose a role for lagging strand replication and its error-prone Pol α tract retention in the formation of these hotspots. I test this hypothesis using Okazaki fragment sequencing data from the yeast Saccharomyces cerevisiae to identify peaks in Okazaki junctions. When these peaks are aligned and orientated, so that the direction of lagging strand replication is uniform, I find a peak in substitution rate immediately downstream of Okazaki junctions, precisely where Pol α tract retention is predicted to occur. Novel binding motifs are identified within the underlying DNA of these junctions that can be assigned to known strong and fast-binding transcription factors, previously implicated in the phasing of nucleosomes, such as Reb1. I show that mutation hotspots adjacent to transcription factor binding sites are a conserved feature of eukaryotic genomes. In the human genome I predict sites of preferential Pol α retention using DNase I hypersensitivity footprint data. We observe that those footprints predicted as germline-specific manifest an elevated mutation signature. I propose that the rapid binding of some transcription factors to DNA following replication is required for nucleosome positioning or other important functions, however this incurs a cost in terms of locally elevated mutation rate adjacent to and within the sequence specific binding site. As a consequence these binding sites are biologically important mutational hotspots whose functional significance has been systematically underestimated by standard measures of sequence constraint.

To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions. To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function. The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA. As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template. Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels. In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.

<p>Abstract</p><p>DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.</p><p>DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.</p><p>In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.</p><p>The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.</p><p>We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).</p><p>Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome. </p><p>We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.</p><p>In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.</p><p>In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program.&#8195;</p> / Dissertation