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.

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.

<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