The GINS complex (a heterotetramer of Sld5, Psf1, Psf2 and Psf3) is a highly conserved DNA replication factor required for the initiation and elongation of DNA replication. GINS is believed to associate with Cdc45 and MCM proteins on replicating DNA. The interaction between GINS and MCM is also conserved in archaea. In my thesis, I explore the subcellular localisation of the GINS complex in relation to the MCM proteins and sites of DNA replication by high-resolution confocal microscopy. For these studies, I generated and carefully validated purified rabbit polyclonal and mouse monoclonal antibodies; these show a specific staining pattern by immunohistochemistry, immunoblotting and immunofluorescence. At high-resolution, all GINS antibodies produced a focal nuclear pattern, similar to that seen for the MCMs. However, confusingly, colocalisation between GINS and MCMs and between the GINS subunits themselves is poor. Investigations are continuing to understand this conundrum. Given the value of MCM proteins as specific and sensitive markers for cancer screening, I investigated whether GINS subunits also have potential diagnostic value. Sld5 and Psf3 expression is restricted to the proliferative compartment in normal tissue, but is found in the majority of cells in a wide range of dysplastic and malignant tissues, including cervix, colon and bladder. In vitro studies of tissue culture cells and cell lysates incubated in urine suggest that Sld5 protein is more stable than Mcm2 in harsh extracellular environments. In an ongoing pilot clinical study of Sld5 protein as a potential biomarker, Sld5 is readily and specifically detectable in the cellular fraction of the samples from prostate and bladder cancer patients. Work is ongoing to evaluate Sld5 protein levels in the supernatant portion of those same urine samples as an easy-to-screen diagnostic/prognostic marker for male urogenital cancers. Owing to their stability, GINS proteins hold promise as independent or complementary markers to the MCM proteins for cancer screening in harsh extracellular environments such as urine.

The accurate replication of the mammalian genome is a complex and logistically challenging process. The entirety of the genome must undergo a single duplication with as little error as possible. This must occur in a coordinated fashion and over suitably short time scale so as to allow timely cellular division within a cell cycle that is typically around 24 hours in a human cell. A great wealth of knowledge already exists describing various aspects of the S phase, during which this replication of the genome occurs. This data has been gathered over a variety of model systems, ranging from inferences from the replicative mechanics of SV40 through to direct observations of replication in mammalian cells.In order integrate this data and determine the value of inferences from different data sources, quantitative models of the mammalian cell S phase are required. This study documents the development of several such models and the exploration of the influences that experimentally determined parameters and different mechanistic theories can have on the behaviour of a simulated S phase. Of particular exploratory interest were the modes of activating replication of replicon clusters, with the aim of simulating experimentally observed dynamics. Additionally, the study also aimed to investigate the variation of replication fork rates and the density of origins of replication, along with the relationship that occurs between the two during both replicational stress and during a normal S phase. Through an iterative series of models, relevant parameters and key theories are sequentially explored so as to better understand the S phase. Particularly influential parameters were identified and studied in detail, with experimental determination where necessary in order to more accurately inform the model system. Conclusions concerning the behaviour of the system and the potential impact of the results were drawn upon the completion of each level of modelling and experimental work.To conclude the study, a linear model simulating the genome of the MRC5 cell line was used to estimate the modes activation of DNA replication along chromosomes in order to recreate experimentally observed replication dynamics. Experimentally determined profiles of replication fork rates and the density of origin firing were also determined for the MRC5 cell line, and were used to populate the model with accurate and appropriate data. Using the model to simulate S phase through a variety of behavioural parameters, realistic S phase dynamics were found to occur through a combination of de novo activation of replicon clusters and a specific probability of neighbour activation by completed clusters. These derived mechanics, when performed on a system correctly parameterised with suitable data, can simulate experimentally observed phenomena. The development of the model highlighted the requirements of data fit for purpose, and the study also stresses the need for critical consideration of inferences made between different model systems.

A replicação do DNA tem início nas origens de replicação que são licenciadas na transição das fases M/G1, pelo complexo de pré-replicação (CPR), e ativadas apenas na fase S. Existem diversas origens de replicação no genoma, mas apenas parte destas origens é disparada em diferentes momentos de S, havendo assim origens early (disparadas no início de S) e late (disparadas mais tardiamente). Em trypanosomas as origens de replicação são reconhecidas por um CPR formado por Orc1/Cdc6 e pelo complexo MCM2-7. Em T. cruzi observamos que existem dois mecanismos diferentes para controlar a replicação do DNA. Durante o ciclo celular da forma epimastigota, as proteínas do CPR são sempre expressas e ligadas ao DNA, mas durante o ciclo de vida Orc1/Cdc6 se liga ao DNA apenas nas formas que replicam, e Mcm7 não é expressa nas que não replicam. Também foi analisado o perfil das forquilhas de replicação em T. brucei utilizando a técnica de SMARD onde vimos que a velocidade da forquilha é semelhante a dos demais eucariontes, além de encontrarmos a primeira origem de replicação late. / The DNA replication starts at the origins of replication, which are licensed at M/G1 transition, by the pre replication complex (PRC), and are activated just at S phase. There are many origins of replication along genome, but some of them are fired at different moments of S phase. So there are early and late origins fired at the beginning or later in S phase, respectively. The PRC of trypanosomes is composed of Orc1/Cdc6 and Mcm2-7. We could observe that in T. cruzi there are two distinct ways to control DNA replication. Whereas in epimastigote cell cycle the PRC are expressed and bound to DNA in all phases, during T. cruzi life cycle Orc1/Cdc6 is bound to DNA only in replicative forms and Mcm7 is absent in the non-replicative forms. We also analyzed the fork profile in T. brucei through SMARD technique. We found that the speed of replication fork is similar from other eukaryotes and that different replication origins are fired every cell cycle. Finally, we found a new origin of replication that is the first late origin described in this organism.

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.

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.

In a previous publication [1] the author described the base rivalry in monotonous DNA sequences and their effect on the DNA repair mechanism. As described in the article, during the monotonous sequence replication, energies appear theoretically to increase with a progressive replication fork up to the quantum mechanical energy level n=2 because of the base rivalry, and these rivalry energies affect the bond strength between the complementary bases. If there is a tautomeric base pair in the replication position where the rivalry energy is large enough, then in this position an irreparable mutation will occur, since the DNA repair mechanism cannot repair that error because too much binding energy. Thus a mutation (caused by base rivalry) can occur only on condition that a transition of a base pair into its tautomeric form is happened. It is remarkable that this transition likewise can occur by the effect of base rivalry energy. The base rivalry - energy which has an effect on a normal base pair provokes a tunnel process in its hydrogen bond, and produces the tautomeric form. After whose replication a different, irreparable base pair develops from the tautomeric base pair, when the rivalry - energy leads into a very strong hydrogen bond. This happens, however, by chance and in the following we will compute the probabilities of such accidental events. The result of these calculations is the equation (32) which could be useful for the theory of evolution and besides for clearing up of virus mutations. It is remarkable that follows from these calculations that the length of DNA increases itself in the course of evolution (section 7).

In the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus−Ter explains its low efficiency in vivo and why contradictory in vitro results from previous studies have led to controversial elucidations of the mechanism. It also provides the first example where the intrinsic heterogeneity in rate of individual replisomes could have different biological outcomes in its communication with double-stranded DNA-binding protein barriers.

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

DNA replication is a fundamental process that is primarily regulated at the initiation step. In higher eukaryotes, the location and properties of replication origins are not well understood. Existing genome-wide approaches to map origins—such as nascent strand abundance mapping, Okazaki fragment mapping, or chromatin immunoprecipitation-based assays—average the behavior of a population of cells. However, due to cell-to-cell variability in origin usage, single molecule techniques are necessary to investigate the actual behavior of a cell. Here, I investigate the feasibility of using three single molecule, genome-wide technologies to map origins of replication. The Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing technology, the BioNano Genomics Irys optical mapping technology, and the Oxford Nanopore Technologies MinION nanopore sequencing technology are promising approaches that can advance our understanding of DNA replication in higher eukaryotes.

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