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.

The final stages of replication fork termination requires the timely and orderly orchestration of catalytic and enzymatic activities. Given the complexity of this process, it is conceivable that the final stages of fork termination is susceptible to problems that could trigger recombination, which could lead to deleterious genomic rearrangements if ectopic homologous sequences are recombined. Using the site-specific RTS1 barrier in fission yeast, I demonstrated that fork termination is generally not a recombinogenic process, and that hyper-recombination-induced by fork blockage at RTS1 is largely a result of replication fork restart. To investigate the actual mechanisms and proteins, which drive and influence recombination at a replication barrier, I studied the MHF proteins, which assist Fml1 in limiting crossovers during double-strand break (DSB) repair and promoting Rad51-mediated recombination at impeded replication forks, and are also components of the constitutive centromere-associated network (CCAN). Intriguingly, structural studies revealed that the MHF can exist as an octamer in vitro. I examined the biological significance of octameric MHF by employing three mutations that disrupt the octamer configuration in vitro. In fission yeast, these mutations cause hypersensitivity to methyl methanesulfonate (MMS), suggesting that the MHF octamer may have a role in DNA repair. One of the “octamerisation” mutants, exhibits greater hypersensitivity to MMS than the other two, and biochemical experiments indicated that this is because it confers an additional defect in MHF’s interaction with Fml1. Further genetic experiments on this mutant suggest that the ability of Fml1 to unwind D-loops depends more critically on its interaction with MHF than fork reversal. Additionally, I showed a synergistic interaction between Dcr1 and MHF, and demonstrated that in the absence of Dcr1, there is a greater need for recombination to tolerate/repair DNA damage. Lastly, I uncovered a novel function for the MHF in controlling the initiation of septation.

The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast. Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.

In response to DNA damage, the ATM and ATR kinases proliferate a signal that is transduced, either directly or via Chk2 and Chk1, to effector proteins, forming the DNA damage response (DDR). The effector proteins delay cell cycle progression, through checkpoints, and activate specific DNA repair mechanisms essential for preserving genome integrity and preventing cancer formation. Bloom's syndrome (BS) patients, which lack the BLM protein show genome instability and have a predisposition to cancer. BLM is phosphorylated by the DDR kinases ATM, ATR and Chk1. These phosphorylation events are essential for BLM to maintain replication fork integrity, preserve the S phase checkpoint and activate BLM to interact with other DDR proteins. In this study I have shown that BLM, isolated from mitotic cells, is phosphorylated on amino acid residue serine 26 (S26). BS cells lacking native BLM, but expressing a variant of BLM protein that cannot be phosphorylated at S26, fail to fully activate the G2/M checkpoint following UV irradiation or treatment with inhibitors of DNA topoisomerase H. Consequently, these cells are more sensitive to killing by these agents than are BS cells expressing wildtype BLM. The Chk1 and Aurora B kinases are able to phosphorylate BLM on S26 in vitro. Moreover, loss of Aurora B kinase activity leads to reduction of S26 phosphorylation in mitotic cells. Cells treated with inhibitors of Aurora B fail to fully active the G2/M checkpoint after UV DNA damage. Taken together, these data suggest, that Aurora B kinase phosphorylates BLM on S26 and that this is required to fully activate the G2/M checkpoint.

Estudou-se o comportamento frente a radiação UV de células humanas XP12RO, deficientes em reparo-excisão de dímeros de pirimidina. Cinéticas de incorporação de precursor radiativo de DNA em tempos crescentes apos a exposição a UV mostraram uma rápida inibição da taxa de síntese, até se alcançar um platô bem abaixo do valor obtido para células não irradiadas. Tanto o tempo para que este platô fosse alcançado quanto o valor basal de síntese obtido dependiam da dose de UV fornecida. Este tempo era compatível com o necessário para que a maquinaria de replicação percorresse a distância média interdímeros esperada para a dose de UV aplicada. Verificou-se também que o DNA recém-sintetizado após UV apresentava um peso molecular e uma taxa de elongação bem menores que nos controles não irradiados, sugerindo todos estes resultados que o dímero se constitue num bloqueio temporário para a replicação de DNA. Utilizando uma metodologia baseada no tratamento do DNA nativo com S1 endonuclease de Aspergillus oryzae, específica para DNA simples-fita, foi possível detectar a existência de lacunas de DNA replicado após UV, lacunas estas que desaparecem gradativamente com o passar do tempo pós-irradiação. DNA não irradiado manteve-se refratário à enzima, nas mesmas condições. A digestão enzimática acarretava o aparecimento de duas populações distintas de DNA, uma de alto peso molecular e outra de peso molecular bem menor, ambas se equivalendo em quantidade. Este fenômeno pôde ser observado em uma ampla faixa de doses de UV, tanto em células XP12RO como em outras linhagens celulares, e mesmo sob condições diversas de proliferação celular. Além disso, o desaparecimento das lacunas, no caso de células de roedor previamente irradiadas com UV, era retardado pela presença de cafeína, um conhecido inibidor de reparo pós-replicação (RPR) nestas linhagens. Foi efetuada uma análise da progressão da forquilha de replicação e da distribuição de lacunas do DNA replicado após UV, através de ensaios enzimáticos combinados com bandeamento de DNA em gradientes isopícnicos de CsCl. Os resultados assim obtidos levaram-nos a considerar um modelo de replicação a partir de molde lesado onde síntese descontínua (3\'-5\') propicia a formação de lacunas, enquanto que síntese contínua (5\'-3\') é retardada temporariamente pela presença da lesão, sem contudo acarretar a formação de descontinuidades físicas no DNA replicado. A mesma metodologia de digestão de DNA com S1 endonuclease permitiu verificar a ocorrência de uma nítida relação causal entre a frequência de lacunas e a frequência correspondente de dímeros, em crescentes doses de UV, sugerindo fortemente que dímeros estão opostos às lacunas no DNA recém sintetizado. Além disso, um tratamento estatístico da cinética de clivagens enzimáticas observada para as lacunas tornou possível calcular a extensão física destas, detectando-se a presença de duas populações distintas, onde 65% correspondem a 1250 nucleotídeos e 35% correspondem a 150 nucleotídeos. Finalmente, foi verificado que DNA recém-sintetizado longos tempos após UV apresenta um drástico declínio da frequência de lacunas, não obstante a frequência de dímeros permanecer essencialmente inalterada. Estes resultados favorecem a hipótese de ocorrer um processo induzido de RPR, o qual permitiria à maquinaria de replicação transpor eficientemente os dímeros presentes, apesar destes não terem propriedades codificadoras. / The synthesis of DNA in human XP12RO cells, deficient in excision repair of pyrimidine dimers was studied. The rate of incorporation of radioactive precursors into DNA was measured at different times after irradiation. The DNA synthesis decreases shortly after irradiation, reaching a lateau whose value and time to be attained was dependent on the UV dose. This time period was the one expected for the replication machinery to coyer the interdimer distance at the UV dose applied. It was also verified that the newly synthesized DNA after UV irradiation presented much smaller molecular weight and elongation rate, when compared with the non-irradiated controls. These results suggest that the dimer imposes a delay to DNA replication machinery. Using a methodology based on the treatment of native DNA with S1-endonuclease from Aspergillus orizae, specific for single-stranded DNA, it was possible to detect gaps in the DNA replicated after UV treatment. Thesee gaps disapeared gradually with time after irradiation. The nonirradiated DNA remained refractory to the enzyme, under the same experimental conditions. The enzymatic digestion originated approximately equal alounts of two distinct double-stranded DNA populations, one of high molecular weight and other of much smaller molecular weight. This fenomenon could be seen on a wide range of UV doses, in XP12RO cells as well as in other cells lines, and did not depend on the particular conditions of cell proliferation. Furthermore, the gap disappearence, in the case of rodent cells previously irradiated with UV, was delayed by the presence of caffeine, a known post-replication repair (PRR) inhibitor in these cell lines. An analysis of the progression of the replication fork and of the distribution of gaps in the DNA replicated after UV irradiation was carried out through enzymatic assays combined with DNA banding in isopicnic CsCl gradients. The results thus obtained led us to consider a model for replication on damaged template, whereby gaps are formed only in the strand replicating opposite the fork movement (3\'-5\'). The strand replicating in the same direction as the fork movement (5\'-3\') is temporarily delayed by the presence of the lesion, without originating gaps in the replicative DNA. The same methodology of DNA digestion with S1-endonuclease permitted us to verify the occurrence of a nitid relationship between the gap frequency and the corresponding dimer frequency, for different doses of UV, strongly suggesting that the dimers are opposite the gaps in the newly-synthesized DNA. Furthermore, an statistical analysis of the dependende of DNA cleavage by S1-endonuclease on the enzyme concentration rendered it possible to calculate the size of the gaps. Two distinct populations were detected, 65% corresponding to 1250 nucleotides and 35% corresponding to 150 nucleotides. Finally, it was verified that the nascent DNA synthesized long periods after UV are essentially free of gaps although the dimer frequency remained almost unaltered. These results favour the hypothesis of the occurrence of an induced process of PRR, which would permit the replication machinery to efficiently bypass the dimers, in spite of the fact that these lesions do not exhibit codifying properties.

Timely completion of DNA replication in each cell cycle is crucial for maintaining genomic integrity. This is often challenged by the presence of various replication fork barriers (RFBs). On collision with a RFB, the fate of the replication fork remains uncertain. In some cases, the integrity of the fork is maintained until the barrier is removed or the fork is rescued by merging with the incoming fork. However, fork stalling can cause dissociation of all of the associated replication proteins (fork collapse). If this occurs, the cell's recombination machinery can intervene to help restart replication in a process called recombination-dependent replication (RDR). Programmed protein-DNA barriers like the Replication Terminator Sequence-1 (RTS1) have been used to demonstrate that replication fork blockage can induce recombination. However, it remains unclear how efficiently this recombination gives rise to replication restart and whether the restarted replication fork exhibits the same fidelity as an origin-derived fork. It is also unknown whether accidental replication barriers induce recombination in the same manner as programmed barriers. In this study, I introduce recombination reporters at various sites downstream of RTS1 to obtain information on both the fidelity and efficiency of replication restart. I find that unlike break induced replication (BIR), the restarted fork gives rise to hyper-recombination at least 75 kb downstream of the barrier. Surprisingly, fork convergence, rather than inducing recombination, acts to prevent or curtail genetic instability associated with RDR. I also investigate a number of genetic factors that have a role in either preventing or promoting genome instability associated with the progression of the restarted fork. To compare RTS1 with an accidental protein-DNA barrier, a novel site-specific barrier system (called MarBl) was established based on the human mariner transposase, Hsmar1, binding to its transposon end. Replication fork blockage at MarBl strongly induces recombination, more so than at RTS1. This appears to be a general feature of accidental barriers as introduction of the E. coli TusB-TerB site-specific barrier in S. pombe gives rise to a similar effect. Here, I compare and contrast accidental barriers with programmed barriers. I observe that there is very little replication restart, if any, at MarBl measured by direct repeat recombination downstream. This points to the fact that accidental barriers do not trigger fork collapse in the same way as programmed RFBs and that the increased recombination that they cause may be a consequence of the inability of replication forks to terminate correctly, owing to the bi-directional nature of the barrier. Several genetic factors are assessed for their impact on MarBl-induced recombination, which further highlights both similarities and differences with RTS1-induced recombination.

Within the past 70 years since the discovery of 5-methylcytosine, we have acquired considerable knowledge about genomic DNA methylation patterns, the dynamics of DNA methylation throughout development, and the enzymatic machinery that establishes and perpetuates genomic methylation patterns. Nonetheless, in the field of epigenetics major questions remain open about the mechanisms of spatiotemporal control that exist to ensure the fidelity of methylation patterns. This thesis aims to decipher the regulatory logic and upstream pathways influencing one of the DNA methyltransferases by leveraging the diverse resources of molecular genetics, biochemistry, and structural biology. The primary subject of my research, DNA methyltransferase 1 (DNMT1), is crucial for maintaining genomic methylation patterns upon DNA replication and cell division. In addition to its C-terminal catalytic domain, mammalian DNMT1 harbors several N-terminal domains of unknown function: a succession of seven glycine-lysine (GK) repeats, resembling histone tails, and two Bromo-Adjacent Homology (BAH) domains that are absent from bacterial DNA methyltransferases. The work I present in this thesis characterizes the role of these hitherto enigmatic domains in regulating DNMT1 activity. In my studies, I found that mutation of the (GK) repeats motif leads to de novo methylation by DNMT1 specifically at paternally imprinted genes. Conventionally, de novo methylation is thought to be undertaken by complete different enzymes, DNMT3A and DNMT3B, whereas DNMT1 is limited to perpetuating the patterns these other methyltransferases had set down. Recombinant DNMT1 had been previously shown to efficiently methylate unmethylated DNA substrate in vitro, but this is the first time its de novo methyltransferase capability has been observed in vivo. Based on these data, I propose a new model in which DNMT1 is the enzyme responsible for laying down de novo methylation patterns at paternally imprinted genes in the male germline, explaining the previously observed non-essential role of other DNA methyltransferases in the establishment of paternal imprints. Furthermore, I demonstrated that acetylation of the (GK) repeats motif inhibits this de novo methyltransferase activity of DNMT1, making this particular motif an essential regulatory platform for controlling the diverse in vivo functions of the enzyme. Though the (GK) repeats motif had previously been proposed to regulate the stability of DNMT1 protein through its interaction with the deubiquitinase USP7, I tested the biological relevance of this interaction and found that USP7 deletion does not alter DNMT1 protein levels. In fact, USP7 appears to play no part in regulating maintenance DNA methylation, as I present evidence that USP7 localization to replication foci is entirely independent of DNMT1. Finally, I demonstrated that the tandem BAH domains of DNMT1 are required for its maintenance methyltransferase activity as they are involved in targeting the enzyme to replication foci during S phase. Based on biochemical data supporting an interaction between DNMT1's BAH1 domain and histones, I propose that this targeting could occur through BAH1's recognition of specific histone modifications, thus providing a potential mechanistic link between maintenance DNA methylation and chromatin markings. This thesis identifies DNMT1 as a novel de novo methyltransferase in vivo and also characterizes the regulatory functions of the enzyme's BAH domains and the (GK) repeats. These results elucidate the multiple regulatory mechanisms within the DNMT1 molecule itself that control its functions in mammalian cells, thereby providing critical insights as to how the DNA methylation landscape takes shape and yielding surprising revelations about the parts that well-studied proteins have to play in this process.

Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer. The ssDNA-binding activity of RPA is primarily mediated by two domains in the RPA1 subunit. I characterized mutant forms of RPA to elucidate the contribution of specific residues in the high affinity DNA binding domains to the cellular function of RPA. These studies enhance the understanding of the properties of RPA that contribute to DNA repair and cellular checkpoints. Mutation of a conserved leucine residue to proline in the high-affinity DNA binding site of RPA (residue L221 in human RPA) has been shown to have a high rate of chromosomal rearrangements in yeast and mice. I characterized the equivalent mutation in human RPA. My studies show that the mutation causes a defect in ssDNA binding and a nonfunctional protein. Combined with the mice studies, the data suggest that haploinsufficiency of RPA causes an increase in DNA damage and in the incidence of cancer. The ssDNA-interactions of the high affinity binding domains in RPA1 are mediated by several residues including four highly conserved aromatic residues. Mutation of these residues had no effect on DNA replication but caused defects in DNA repair pathways. I conclude that DNA intermediates in different DNA metabolic pathways require different RPA binding functions and that the aromatic residues are indispensable for binding in DNA repair. These studies illustrate that different DNA metabolic pathways have distinct requirements for RPA function. A decrease in binding to ssDNA of any length has specific consequences in vivo. These data also demonstrate that a single mutation in RPA in a residue that does not even contact ssDNA can result in a non-functional RPA complex. I conclude that even a modest decrease in RPA protein levels is not compatible with long term cell survival. Taken together, these studies highlight the importance of proper regulation of RPA protein levels and its ssDNA binding affinity to proper maintenance of the integrity of the genome.

In order to maintain genetic stability in eukaryotes, tight regulation of the relative sizes of deoxyribonucleoside triphosphate (dNTP) levels inside the cell is essential for optimal fidelity of DNA replication. Ribonucleotide reductase (RNR) is the enzyme responsible for proportional production of DNA precursors. Studies on regulation of this enzyme, the focus of this thesis, are important because mutations affecting RNR control mechanisms result in dNTP pool imbalance, thus promoting mutagenesis. By using mouse RNR as a model for mammalian forms of the enzyme, three major factors--allosteric effectors, rNDP substrate concentrations, and hypoxic conditions--that influence the substrate specificity of RNR have been investigated. Allosteric regulation has been studied by the four-substrate assay, which permits simultaneous monitoring of the four reactions catalyzed by this enzyme in one reaction mixture. Individual dNTPs affect the four activities differentially in a concentration-dependent manner with discrete effects of dTTP and dGTP on reduction of ADP and GDP, respectively. Ribonucleoside diphosphate (rNDP) substrate concentrations are equally important, as their variations lead to different product ratios. Results from nucleotide binding assays indicate that rNDPs directly influence binding of dNTP effectors at the specificity site, one of the two classes of allosteric sites, whereas ADP has an indirect effect, displacing other substrates at the catalytic site and consequently removing effects of those substrates upon dNTP binding. Hence, this is the first evidence of a two-way communication between the catalytic site and the specificity site. Oxygen limitation also plays an important role in controlling the enzyme specificity. Reactivation of the enzyme at different oxygen tensions, after treatment of the enzyme with hydroxyurea (HU) followed by removal of HU, reveals a distinct sensitivity of GDP reductase to low 0��� levels. Although the basis for specific inhibition of GDP reduction remains to be determined, some possibilities have been ruled out. This research proves that in addition to allosteric regulation by nucleoside triphosphates, mouse RNR is also controlled by other factors. Since these components can simultaneously exert their effects upon enzyme specificity, complex regulatory patterns of RNR to provide a proportional supply of the DNA building blocks in vivo are suggested. / Graduation date: 2003

Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase α (Pol α) synthesize short primers required for DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) to carry out the bulk synthesis. The role of Pol δ and Pol ε at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ε has at the replication fork, compare the biochemical properties of Pol δ and Pol ε, and to study the function of the second largest and essential subunit of Pol ε, Dpb2. To identify where Pol ε replicates DNA in vivo, a strategy was taken where the active site of Pol ε was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ε proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol ε participates in the synthesis of the leading strand. Pol δ and Pol ε are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol ε was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol δ was much less sensitive to uncoated ssDNA. The processivity of Pol δ was stimulated to a much higher degree by PCNA than of Pol ε. In presence of PCNA the processivity of Pol δ and Pol ε was comparable. In contrast, Pol ε was approximately four times slower than Pol δ when replicating a single-primed circular template in the presence of all accessory proteins and an excess of polymerase. The biochemical characterization of the system suggests that Pol ε and Pol δ are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol ε onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand. The essential gene DPB2 encodes for the second largest subunit of Pol ε. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Polε, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol ε (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol ε. We propose that Dpb2 is not essential for the enzyme activity of Pol ε.