In Saccharomyces cerevisiae, RTT107 (ESC4, YHR154W) encodes a BRCT-domain protein that is important for recovery from DNA damage during S phase. I have found that Rtt107 forms a complex with the Slx1/Slx4 structure-specific nuclease. Deletion of SLX4 confers many of the same phenotypes observed in rtt107∆, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage, suggesting that Slx4 and Rtt107 function in concert. These defects are not shared by Slx1 indicating that the function of Slx4 and Slx1 in the DNA damage response is not entirely overlapping. Furthermore, I found that Slx4 regulates the phosphorylation of Rtt107 by the checkpoint kinase Mec1. The phenotypes conferred by deletion of RTT107 and the spectrum of its synthetic genetic interactions indicates that Rtt107 may function at stalled replication forks. I have shown that Rtt107 is recruited to chromatin in the presence of DNA damaging agents that cause DNA replication forks to stall. Recruitment of Rtt107 to chromatin requires Rtt109, an acetyltransferase, and the cullin Rtt101, but is not dependent on Slx4 or the checkpoint kinases. Rtt109 acetylates histone H3 on lysine 56 (H3-K56), yet recruitment of Rtt107 to chromatin does not require acetylation of H3-K56, indicating that Rtt109 may have additional targets. Chromatin immunoprecipitation indicates that the sites of Rtt107 binding correspond to regions at or near stalled replication forks throughout the genome. I propose that Rtt107 acts in the recovery from DNA damage by localizing to stalled replication forks and acting as a scaffold for assembly of DNA damage response proteins, ultimately promoting replication fork restart.

The Dbf4 protein is involved in the initiation of DNA replication, in complex with Cdc7 kinase, and also plays a role in the intra-S-phase checkpoint response via an interaction with Rad53 in Saccharomyces cerevisiae. The Dbf4 protein has three highly conserved motifs, called the N, M and C motifs. In view of the fact that a comprehensive analysis of the roles of the three motifs in the initiation of DNA replication and checkpoint response was not previously available, this study was, therefore, conducted. The objectives of the study were: (1) to assess the function of the three conserved motifs, with respect to their essentiality for cell viability, (2) to determine their roles in mediating interactions with other proteins (i.e. Cdc7, Orc2, Mcm2) involved in the initiation of DNA replication and with Rad53 in the intra-S-phase checkpoint response, and (3) to obtain the three-dimensional structure of the Dbf4 N-motif by X-ray crystallography. The Dbf4 N-motif was found to be nonessential for cell viability, mediates the interaction between Dbf4 and Rad53, and as well as the interaction with Orc2. A mutant lacking the N-motif (dbf4N), was found to have a growth defect and was hypersensitive to the genotoxic agents: hydroxyurea (HU) and methyl methane sulfonate (MMS), suggesting that a disruption in the intra-S-phase checkpoint occurred because of an abrogated Dbf4-Rad53 interaction. Double point mutation of two threonine residues of the N-motif (threonines 171 and 175) to alanines also caused an abrogated Dbf4-Rad53 FHA1 domain interaction. The Dbf4 M-motif was found to be essential for cell viability and mediates the interaction between Dbf4 and Mcm2. A single proline to leucine point mutation at amino acid residue 277 conferred resistance to HU and MMS and caused disrupted Dbf4-Mcm2 and Dbf4-Orc2 interaction, while Dbf4-Rad53 interaction was maintained. Thus, the alteration of the M-motif may facilitate the role of Dbf4 as a checkpoint target. The Dbf4-C motif was also found to be essential for cell viability. Deletion and point mutations to the C-motif affected the interactions between Dbf4 and Rad53, Orc2, Mcm2 and also with Mcm4. Attempts were also made to obtain the three-dimensional structure of Dbf4, using X-ray crystallography methods. The work presented here represents a thorough functional analysis of the three conserved domains of Dbf4 in Saccharomyces cerevisiae. These results can be used as a baseline for further research involving higher eukaryotic organisims, including humans. This is particularly of relevance in light of recent evidence demonstrating an overexpression of the human Dbf4 orthologue overexpression as a cancer phenotype in human cancer cells.

The origin recognition complex (Orc1-6) plays a fundamental role in the initiation of DNA replication by binding replication origins throughout the budding yeast cell cycle. ORC acts as a scaffold for the assembly of the pre-replicative complex (pre-RC) factors Cdc6, Cdt1 and a replicative helicase, the minichromosome maintenance (MCM2-7) complex in G1 phase. Upon assembly, origins are then said to be “licensed” for DNA replication. Previous models of pre-RC assembly and function have predicted that once MCMs have been loaded onto chromatin, ORC and Cdc6 are no longer required for DNA replication. In contrast, data from our lab strongly suggest a role for Orc6 in the maintenance of MCMs after pre-RC formation. Orc6 was found to be required for the chromatin maintenance of Mcm2 and more specifically, chromatin immunoprecipitation (ChIP) analysis demonstrated that Orc6 was necessary for the continued origin association of MCM proteins in late G1 at the early-firing origin ARS1, and the late-firing origin ARS609. It was also determined that after destabilization in the absence of Orc6, the pre-RC could be reassembled and facilitate DNA replication in late G1 after Orc6 expression had been turned back on. Interestingly, the clamp loading protein Cdc6 was also discovered to be essential to the maintenance of MCM proteins on bulk chromatin and at ARS1.

The red algae (Rhodophyta) is a monophyletic phylum and is comprised of seven classes including the Florideophyceae. The class monophyletic Florideophyceae is postulated to have diverged from the class Bangiophyceae and contains over 32 orders including the freshwater Batrachospermales and Thoreales. Classifications within these orders as well as the class are based predominantly on female reproductive characters and vegetative morphology. The order Batrachospermales contains a number of families and genera with the genus Batrachospermum being the largest with eight recognized sections. The order Thoreales was once considered a genus within the order Batrachospermales but is currently recognized as an autonomous order. Due to the cryptic nature of the genera, particularly Batrachospermum, the Morphological Species Concept has proven to be limiting in the classification at the species level. This study examines the usefulness of the mitochondrial gene encoding cytochrome c oxidase subunit I (COI) in delimiting species within the orders Batrachospermales and Thoreales from several countries spanning three continents and comparing this data to a parallel analysis of the gene encoding the large subunit of the chloroplast enzyme Ribulose 1, 5-bisphosphate carboxylase/oxygenase (rbcL). Sequence data and phylogenetic analysis illustrates possible delineation among species using the COI marker. Distinct clades of sections Batrachospermum, Setacea, Virescentia, Turfosa, Contorta, Gonimopropagulum and Aristata were observed from various geographic locations; as well as clades of genus Sirodotia, Tuomeya Lemanea, Paralemanea and Thorea. The present study proposes the elevation of the current recognized infrageneric sections to the status of genus based on both COI and rbcL genes sequence data. In addition, very few clades appeared to reflect any biogeographic trends.

The Dbf4 protein is involved in the initiation of DNA replication, in complex with Cdc7 kinase, and also plays a role in the intra-S-phase checkpoint response via an interaction with Rad53 in Saccharomyces cerevisiae. The Dbf4 protein has three highly conserved motifs, called the N, M and C motifs. In view of the fact that a comprehensive analysis of the roles of the three motifs in the initiation of DNA replication and checkpoint response was not previously available, this study was, therefore, conducted. The objectives of the study were: (1) to assess the function of the three conserved motifs, with respect to their essentiality for cell viability, (2) to determine their roles in mediating interactions with other proteins (i.e. Cdc7, Orc2, Mcm2) involved in the initiation of DNA replication and with Rad53 in the intra-S-phase checkpoint response, and (3) to obtain the three-dimensional structure of the Dbf4 N-motif by X-ray crystallography. The Dbf4 N-motif was found to be nonessential for cell viability, mediates the interaction between Dbf4 and Rad53, and as well as the interaction with Orc2. A mutant lacking the N-motif (dbf4N), was found to have a growth defect and was hypersensitive to the genotoxic agents: hydroxyurea (HU) and methyl methane sulfonate (MMS), suggesting that a disruption in the intra-S-phase checkpoint occurred because of an abrogated Dbf4-Rad53 interaction. Double point mutation of two threonine residues of the N-motif (threonines 171 and 175) to alanines also caused an abrogated Dbf4-Rad53 FHA1 domain interaction. The Dbf4 M-motif was found to be essential for cell viability and mediates the interaction between Dbf4 and Mcm2. A single proline to leucine point mutation at amino acid residue 277 conferred resistance to HU and MMS and caused disrupted Dbf4-Mcm2 and Dbf4-Orc2 interaction, while Dbf4-Rad53 interaction was maintained. Thus, the alteration of the M-motif may facilitate the role of Dbf4 as a checkpoint target. The Dbf4-C motif was also found to be essential for cell viability. Deletion and point mutations to the C-motif affected the interactions between Dbf4 and Rad53, Orc2, Mcm2 and also with Mcm4. Attempts were also made to obtain the three-dimensional structure of Dbf4, using X-ray crystallography methods. The work presented here represents a thorough functional analysis of the three conserved domains of Dbf4 in Saccharomyces cerevisiae. These results can be used as a baseline for further research involving higher eukaryotic organisims, including humans. This is particularly of relevance in light of recent evidence demonstrating an overexpression of the human Dbf4 orthologue overexpression as a cancer phenotype in human cancer cells.

The origin recognition complex (Orc1-6) plays a fundamental role in the initiation of DNA replication by binding replication origins throughout the budding yeast cell cycle. ORC acts as a scaffold for the assembly of the pre-replicative complex (pre-RC) factors Cdc6, Cdt1 and a replicative helicase, the minichromosome maintenance (MCM2-7) complex in G1 phase. Upon assembly, origins are then said to be “licensed” for DNA replication. Previous models of pre-RC assembly and function have predicted that once MCMs have been loaded onto chromatin, ORC and Cdc6 are no longer required for DNA replication. In contrast, data from our lab strongly suggest a role for Orc6 in the maintenance of MCMs after pre-RC formation. Orc6 was found to be required for the chromatin maintenance of Mcm2 and more specifically, chromatin immunoprecipitation (ChIP) analysis demonstrated that Orc6 was necessary for the continued origin association of MCM proteins in late G1 at the early-firing origin ARS1, and the late-firing origin ARS609. It was also determined that after destabilization in the absence of Orc6, the pre-RC could be reassembled and facilitate DNA replication in late G1 after Orc6 expression had been turned back on. Interestingly, the clamp loading protein Cdc6 was also discovered to be essential to the maintenance of MCM proteins on bulk chromatin and at ARS1.

The red algae (Rhodophyta) is a monophyletic phylum and is comprised of seven classes including the Florideophyceae. The class monophyletic Florideophyceae is postulated to have diverged from the class Bangiophyceae and contains over 32 orders including the freshwater Batrachospermales and Thoreales. Classifications within these orders as well as the class are based predominantly on female reproductive characters and vegetative morphology. The order Batrachospermales contains a number of families and genera with the genus Batrachospermum being the largest with eight recognized sections. The order Thoreales was once considered a genus within the order Batrachospermales but is currently recognized as an autonomous order. Due to the cryptic nature of the genera, particularly Batrachospermum, the Morphological Species Concept has proven to be limiting in the classification at the species level. This study examines the usefulness of the mitochondrial gene encoding cytochrome c oxidase subunit I (COI) in delimiting species within the orders Batrachospermales and Thoreales from several countries spanning three continents and comparing this data to a parallel analysis of the gene encoding the large subunit of the chloroplast enzyme Ribulose 1, 5-bisphosphate carboxylase/oxygenase (rbcL). Sequence data and phylogenetic analysis illustrates possible delineation among species using the COI marker. Distinct clades of sections Batrachospermum, Setacea, Virescentia, Turfosa, Contorta, Gonimopropagulum and Aristata were observed from various geographic locations; as well as clades of genus Sirodotia, Tuomeya Lemanea, Paralemanea and Thorea. The present study proposes the elevation of the current recognized infrageneric sections to the status of genus based on both COI and rbcL genes sequence data. In addition, very few clades appeared to reflect any biogeographic trends.

The interest in using gene therapy to target a variety of both inherited and acquired diseases has intensified over the last two decades. Because free DNA is easily degraded by serum nucleases in the bloodstream, the need for developing carrier systems that can compact and protect the DNA was quickly realized. Viral vector systems were some of the earliest carriers used, primarily because of the ease with which such systems can infect host cells. However, difficulties experienced when using viral vectors, including immunogenicity and the potential for genetic recombinations, forced researchers to design alternative delivery strategies. Non-viral vectors offer one alternative to overcome this dilemma. In addition to avoiding the biological problems experienced using viral carriers, non-viral vectors also offer the potential for large-scale production. Dendrimers are one non-viral carrier that has shown appreciable ability to deliver DNA into cells, a process called transfection. In the past, triazine dendrimers have shown biocompatibility, and the ability to synthesize these structures to contain cationic charges on the surface makes these structures potentially suitable for transfection studies. In this study, a small library of triazine dendrimers was synthesized in an attempt to understand how variations to both the periphery and core of triazine dendrimers affect the transfection efficiency of these dendriplexes. In the first subset of structures, a common core was used and various peripheral groups were appended to the dendrimer surface. The physicochemical and biological data, obtained in collaboration with Thomas Kissel at Philipps-Universitat Marburg, showed that the surface groups have a notable affect on transfection efficiency. Dendrimers with a higher amine number and neutral surface groups show high DNA binding affinity and higher transfection efficiency. In the second subset of dendrimers, variations to the core showed that transfection efficiency is improved both by increasing generation number and dendrimer flexibility. With this data in hand, triazine dendrimers with both higher generation number and higher flexibility have been synthesized. Two different triazine linker groups, trimethylene-dipiperidine and polyglycoldiamine, have been used. These structures will be evaluated to determine if increasing both flexibility and generation number together can further improve transfection efficiency.

DNA molecules stretching and deformation in hydrodynamic flow field has been investigated for many years with various experimental and theoretical methods. This research was performed experimentally in hybridized microchannels made of poly(dimethylsiloxane)(PDMS) and glass. We will directly measure the hydrodynamic stretching DNA molecules in elongation flow by CLSM (Confocal Laser Scanning Microscope). The deformation of YOYO-labeled lambda phage DNA molecules in flows was visualized with CLSM. The relation between DNA contour length and relaxation time in different models of stretching flows was also be presented. Furthermore, the distribution of stretching DNA molecules in microchannels was derived to analyze and compared with the DNA migration velocity and stress relaxation modulus.

Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.