Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 98-115).

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains x, 231 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 188-203).

<p>Every living cell faces a multitude of DNA threats in its lifetime because damage to DNA is intrinsic to life itself. A double-strand break (DSB) is the most cytotoxic type of DNA damage and is a potent inducer of chromosomal aberrations. Defects in DSB repair are a major driver of tumorigenesis and are associated with numerous developmental, neurological and immunological disorders. To counteract the deleterious effects of DSBs, organisms have evolved a homologous repair (HR) mechanism that is highly precise. The key to its error-free nature lies in its use of a homologous template in restoring the DSB and its preferential occurrence during late S and G2 phase of the cell cycle when identical sister chromatids are available as templates for repair. However, HR can also engage homologous chromosomes and ectopic substrates that share homology, resulting in mitotic loss-of-heterozygosity (LOH) and unwanted chromosomal aberrations. In this case, understanding of the underlying mechanisms and molecular factors that influence accurate sequence transfer and exchange between two homologous substrates becomes crucial. </p><p>The focus of this dissertation is examination of the genetic factors and molecular processes occurring at early intermediate steps (DNA end resection and DNA synthesis) of mitotic recombination in Saccharomyces cerevisiae. To model DSB repair, we established a unique plasmid-based assay with a small 8-base pair (bp) gap in the middle of an 800-bp plasmid substrate. To delineate the molecular structures of strand exchange intermediates during HR, we used a 2% diverged plasmid substrate relative to a chromosomal repair template to generate mismatch-containing heteroduplex DNA (hetDNA) intermediates. The assay was performed in a mismatch repair (MMR)-defective background allowing hetDNA to persist and to segregate into daughter cells at the next round of replication. Unexpectedly, even when MMR was inactivated, sequence analysis of the recombinants revealed patches of gene conversion and restoration reflecting mismatch correction within hetDNA tracts. We showed that, in this system, MMR and nucleotide excision repair (NER) correct mismatches via two different mechanisms. While mispairing of nucleotides triggers MMR, NER is recruited by the subtle 6-methyladenine mark on the plasmid substrate, leading to coincident correction of mismatches. The methylation marks on the plasmid were acquired from the bacterial host’s native restriction-modification system during plasmid propagation. </p><p>Formation of hetDNA occurs when a plasmid substrate engages the chromosomal template for repair, forming a D-loop intermediate. D-loop extension requires DNA synthesis by DNA polymerase/s. Translesion synthesis (TLS) polymerases have been implicated in HR in both chicken DT40 cells and fruit fly, but not in yeast. This class of polymerases is known for its low fidelity due to a lack of exonuclease domain and is commonly used for lesion bypass and in extending ends with mismatches. We reported for the first time a requirement of Polζ-Rev1 and Polη (TLS polymerases in S. cerevisiae) for completing gap repair. Moreover, gap-repair efficiency suggested that these two polymerases function independently. We concluded that TLS polymerases are involved in either extending the invading 3’ end and/or in the gap-filling process that completes recombination. </p><p>DNA resection of a DSB serves as a primary step to generate a 3’ single-stranded DNA (ssDNA) for subsequent homologous template invasion, but this process has mostly been studied in the absence of a repair template or when downstream HR steps are disabled. To analyze the individual contributions of identified nucleases to DSB resection in the context of repair, we established a chromosomal assay; the substrate size was increased to 4 kilobases (kb) and 85 SNPs were present at ~50 bp intervals. In this chromosomal assay, resection and DNA synthesis influence the length of hetDNA tracts in the final recombinants, allowing these two steps to be analyzed. We specifically focused on synthesis-dependent strand annealing (SDSA) events, where hetDNA reflects DNA synthesis and extent of resection. Our main conclusions are as follows. DNA end resection on the annealing end of NCO products generated by SDSA is not as extensive as one might expect from resection measured in single-strand annealing (SSA) assays. In addition, although the two long-range resection pathways (Sgs1-Dna2 and Exo1) can support recombination in a redundant manner, hetDNA was significantly reduced upon loss of either. End processing of DSBs is predominantly 5’ to 3’, but we also observed loss of sequences (greater than 8 nt but less than 40 nt) at the 3’ termini. We have tested and ruled out the involvement of Mre11 and Polε proofreading activity. Lastly, Pol32 functions as a subunit of Polδ to promote extensive repair synthesis during SDSA. hetDNA tract lengths were significantly shorter in the absence of the Pol32 subunit of Polδ, providing direct evidence that Polδ extends the invading end during HR. Together, this work advances our understanding of how resection nucleases and DNA polymerase/s function to regulate mitotic recombination outcome and influence the molecular patterns of NCOs.</p> / Dissertation

Mismatch repair corrects errors made during DNA replication and inactive mismatch repair is associated with Lynch Syndrome and sporadic cancer. Genome replication in eukaryotes is accompanied by chromatin formation. The first step in chromatin establishment is nucleosome assembly, that starts with histone tetramer deposition. It is not clear how three important cellular processes: genome replication, mismatch repair and nucleosome assembly are coordinated. Here we analyzed human mismatch repair in the presence of histone deposition in a reconstituted system. We showed that mismatch repair factor inhibits nucleosome assembly on the DNA region with the replicative error. Such a mechanism is important, since in this way DNA with errors remains accessible for mismatch repair system to perform the repair. The DNA synthesis step in mismatch repair is performed by DNA polymerase. Eukaryotes possess two major replicative DNA Polymerases: DNA Polymerase delta and DNA Polymerase epsilon. DNA polymerase delta is involved in mismatch repair. However, it was unknown whether DNA polymerase epsilon can also work in mismatch repair. Here we analyzed human mismatch repair with DNA Polymerase delta and DNA Polymerase epsilon in the environment of histone deposition. Our results indicated that repair activity with both polymerases was activated by histone deposition. Here it was first shown that human DNA Polymerase epsilon performs DNA synthesis during mismatch repair in vitro. Importantly, recent studies have revealed association of Polymerase epsilon mutations with cancer. Since our data showed activity of DNA Polymerase epsilon in mismatch repair, a possible tumor development mechanism may involve inactivation of mismatch repair due to Polymerase epsilon mutations. Overall, our study expanded the understanding of the mechanism of human mismatch repair in the chromatin environment.

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

Abstract DNA replication is a process in which a cell duplicates its genome before cell division, and must proceed accurately and in organized manner to guarantee maintenance of the integrity of the genetic information. DNA polymerases are enzymes that catalyse the synthesis of the new DNA strand by utilizing the parental strand as a template. In addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also needed in other processes like DNA repair and DNA recombination. The DNA polymerase is an essential DNA polymerase in eukaryotes and is required for chromosomal DNA replication. It has also been implicated in DNA repair, recombination, and in transcriptional and cell cycle control. The regulation of the human enzyme was explored by analysing its expression, phosphorylation and protein-protein interactions. Expression of both the A and B subunits of the human DNA polymerase ε was strongly growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA levels were up-regulated at least 5-fold. In actively cycling cells, however, the steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in G1/S phase. The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107 was demonstrated in vitro. A down-regulation element, located immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription factors. A model of the promoter function is presented. Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Cut5, Drosophila melanogaster Mus101 and the human Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose expression is induced at the G1/S border and is required for chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci, which do not represent sites of ongoing DNA replication. However, if DNA is damaged or replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating cell nuclear antigen (PCNA) containing foci that represent stalled replication forks. Finally, phosphorylation of DNA polymerase ε was described and at least three immunologically distinct and differentially phosphorylated forms were shown to exist. Phosphorylation is on serine and threonine residues and shows a cell cycle dependent fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1 co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.

Abstract Analysis of the primary structure of DNA polymerase ε B subunit defined similarities to B subunits of eukaryotic DNA polymerases α, δ and ε as well as the small subunits of DNA polymerase DI of Euryarchaeota. Multiple sequence alignment of these proteins revealed the presence of 12 conserved motifs and defined a novel protein superfamily. The members of the B subunit family share a common domain architecture, suggesting a similar fold, and arguing for a conserved function among these proteins. The contribution of human DNA polymerase ε to nuclear DNA replication was studied using the antibody K18 that specifically inhibits the activity of this enzyme in vitro. This antibody significantly inhibited DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei, but did not inhibit SV40 DNA replication in vitro. These results suggest that the human DNA polymerase ε contributes substantially to the replicative synthesis of DNA and emphasises the differences between cellular replication and viral model systems. The human DNA polymerases ε and δ were found capable of gap-filling DNA synthesis during nucleotide excision repair in vitro. Both enzymes required PCNA and the clamp loader RFC, and in addition, polymerase δ required Fen-1 to prevent excessive displacement synthesis. Nucleotide excision repair of a defined DNA lesion was completely reconstituted utilising largely recombinant proteins, only ligase I and DNA polymerases δ and ε provided as highly purified human enzymes. This system was also utilised to study the role of the transcription factor II H during repair. Human non-homologous end joining of model substrates with different DNA end configurations was studied in HeLa cell extracts. This process depended partially on DNA synthesis as an aphidicolin-dependent DNA polymerase was required for the formation of a subset of end joining products. Experiments with neutralising antibodies reveal that DNA polymerase α but not DNA polymerases β or ε, may represent this DNA polymerase activity. Our results indicate that DNA synthesis contributes to the stability of DNA ends, and influences both the efficiency and outcome of the end joining event. Furthermore, our results suggest a minor role of PCNA in non-homologous end joining.

In studies of enzyme kinetics, reaction time courses are often condensed into a single set of initial rates describing the rate at the start of the reaction. This set is then analysed with the Henri-Michaelis-Menten equation. However, this process necessarily removes information from experimental data and diminishes its statistical significance due to a reduction of available data points. Further, if the examined system does not approach steady-state rapidly, the application of the steady-state-assumption can lead to flawed conclusions. Here, the analysis of two complex enzyme systems by numerical integration of kinetic rate equations is demonstrated. DNA polymerase catalyses the synthesis of DNA in a reaction that involves two substrates, DNA template and dNTP, both of which are highly heterogeneous in nature. The tight binding of DNA to DNA polymerase and its polymer properties prohibit the application of the initial-rate approach. By combining an explicit DNA binding step with a steady-state dNTP incorporation on a template of finite length, the DNA binding parameters and the dNTP incorporation steady-state parameters were estimated from processive polymerisation data in a global regression analysis. This approach is described in Chapter 2 and the results are in good agreement with previously published values. Further properties were investigated in studies of the temperature dependence and solvent isotope dependence of the kinetics. The processive polymerisation of DNA template was monitored using the fluorophore PicoGreen in a simple and inexpensive method described in Chapter 3. The catalytic cycle of ethanolamine ammonia lyase involves the homoloysis of the Co-C bond within the intrinsic B12 cofactor. This homolysis results in the formation of a Co(II)-adenosyl radical intermediate, which can be monitored using stopped-flow spectroscopy. The stopped-flow transients observed for EAL and related enzymes have long been difficult to analyse and interpret, possibly due to rapid methyl group rotation on the substrate. In Chapter 4 of this thesis we were able to rationalise this behaviour using numerical integration of the rate equations of a branched 16-state-kinetic model to fit stopped-flow transients in a global regression analysis. We were able to determine some intrinsic rate constants, and showed that the initial hydrogen atom transfer step is unlikely to have an inflated primary kinetic isotope effect, despite previous claims. More generally, this study demonstrates that the numerical integration analysis used here is likely to be applicable to a broad range of enzyme reaction kinetics.

DNA damage blocks replication by classical DNA polymerases, those that replicate nondamaged DNA during normal DNA replication and repair, by altering the geometry of the DNA. Consequently, translesion synthesis, the replication of damaged DNA, is catalyzed by non-classical DNA polymerases, which are capable of accommodating the inherent distorted geometry of damaged DNA. The yeast Rev1 protein (Rev1p) specifically catalyzes the incorporation of cytosine opposite template guanine and several types of DNA damage utilizing a unique mechanism of nucleotide selection whereby the sidechain of Arg-324 acts as the template by forming hydrogen bonds with the incoming cytosine. To better understand the impact of this protein-template-directed mechanism on nucleotide incorporation, I carried out pre-steady-state kinetic studies with Rev1p. Interestingly, I found that Rev1p's specificity for incorporating cytosine is achieved solely at the initial nucleotide-binding step. In this respect, Rev1p differs from all previously investigated DNA polymerases. Based on these findings and on structures of another enzyme, MutM, I suggest possible structures for complexes of Rev1p with the other incoming nucleotides. DNA polymerase ζ, encoded by the REV3 gene, functions in the error-prone replication of a wide range of DNA lesions by extending from nucleotides incorporated opposite template lesions by other polymerases. Here I describe genetic and biochemical studies of five yeast DNA polymerase ζ mutant proteins. Four mutant proteins do not complement the rev3Δ mutation, and these proteins have significantly reduced or no polymerase activity relative to the wild-type protein. However, the K1061A protein partially complements the rev3Δ mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased ability to distinguish between correct and incorrect substrates (increased fidelity and decreased misextension ability). These findings have important implications for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions. Additionally, I genetically characterized 21 mutant proteins, which may also affect the substrate specificity of this enzyme. The P962L, L1054A, T1063A, and G1215A mutant proteins were partially capable of complementing the rev3Δ mutation and are candidates for biochemical characterization, as they may have altered substrate specificity.

Novel biocatalysts are highly demanded in the white biotechnology. Hence, the development of highly stable and enantioselective biocatalysts with novel functionalities is an ongoing research topic. Here, an osmium ligating single-site ArM was created based on the biotinstreptavidin technology for the dihydroxylation of olefins. For the creation of the artificial catalytic metal center in the streptavidin (SAV) cavity, efficient osmium tetroxide (OsO4) chelating biotin-ligands were created. The unspecific metal binding of the host scaffold was diminished through genetical and chemical modification of the host protein. The created single-site OsO4 chelating ArM was successfully applied in the asymmetric cyclopropanation, revealing a stable and tunable catalytic hybrid system for application. The structural analysis of protein-ligand complexes is essential for the advanced rational design and engineering of artificial metalloenzymes. In previous studies, a SAV-dirhodium ArM was created and successfully applied in the asymmetric cyclopropanation reaction. To improve the selectivity of the SAV-dirhodium complex, the structural location of the organometallic complex in the SAV cavity was targeted and small-angle x-ray scattering (SAXS) was used to obtain the structural information. The SAXS analysis revealed valuable information of the molecular state of the complexes; hence, the method proved to be useful for the structural analysis of protein-ligand interactions. The discovery of novel enzymes from nature is still the major source for improved biocatalysts. One of the most important enzymes used in the molecular biology are DNA polymerases in PCR reactions. The halothermophilic brine-pool 3 polymerase (BR3 Pol) from the Atlantis II Red Sea brine pool showed optimal activities at 55 °C and salt concentrations up to 0.5 M NaCl, and was stable at temperatures above 95 °C. The comparison with the hyperthermophilic KOD polymerase revealed the haloadaptation of BR3 Pol due to an increased negative electrostatic surface charge and an overall higher structural flexibility. Engineered chimeric KOD polymerases with swapped single BR3 Pol domains revealed increased salt tolerance in the PCR, showing increased structural flexibility and a local negative surface charge. The understanding of the BR3 Pol haloadaptation might enable the development of a DNA polymerase tailored for specific PCR reactions with increased salt concentrations.