Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Junghuei Chen, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis supervisor: Robert E. Malone. Includes bibliographic references (p. 258-279).

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Mar. 5, 2009). PDF text: 78 p. : ill. (some col.) ; 8.43 Mb. UMI publication number: AAT 3330302. Includes bibliographical references. Also available in microfilm and microfiche formats.

Meiosis is the process by which sexually-reproducing organisms ensure that precisely half a chromosome set is passed from each parent to the following generation; this circumvents the doubling of the genome that would otherwise occur upon fertilisation. Meiosis occurs via a single round of DNA replication followed by two successive chromosome segregation events. In the first segregation, homologous chromosomes align and become physically linked through the process of meiotic recombination, which is crucial for the accurate segregation of homologous chromosomes. During the second round of segregation, sister chromatids are segregated to produce four haploid daughter cells. Failure to physically tether homologous chromosomes to each other through meiotic recombination can result in the aberrant segregation of homologous chromosomes, which can cause hereditary diseases (aneuploidies) and miscarriages in humans. Meiotic recombination also shuffles alleles of the parental chromosomes, which is crucial for evolution. The study of meiotic recombination, and its regulation, is thus paramount for our understanding of how genetic diversity is generated within populations. The work in this thesis has helped characterise factors, both genetic and environmental, that modulate meiotic recombination in the fission yeast, Schizosaccharomyces pombe. Here, I identify temperature as a major determinant of meiotic recombination outcome; when meiosis is performed at 16°C, significant reductions in meiotic recombination outcome are observed relative to meiosis performed at higher temperatures. Additionally, I present genetic and cytological evidence that the strand resection and strand invasion steps of meiotic recombination are impaired at 16°C relative to higher temperatures, but that double strand break levels appear not to be influenced by temperature. I have also characterised several novel genes predicted to be involved in meiotic recombination, and explored the genetic relationship between several genes already known to be crucial in modulating meiotic recombination. Finally, I have laid the foundations for a future project aiming to map the meiotic recombination landscape across the entire S. pombe genome.

The role of the T7-induced exonuclease (gene 6) in recombination was studied using both molecular and genetic techniques. In the molecular method the fate of parental DNA during parent-to-progeny recombination was examined. A comparison of infections with T7⁺, T7am6 (amber gene 6), or T7ts6 (temperature sensitive gene 6) under permissive and nonpermissive conditions was made. CsCl density gradient analysis of replicative DNA indicated that the T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Analysis of denatured replicative DNA by CsCl density gradient centrifugation indicated that the exonuclease also may be needed for a limited amount of covalent repair of recombinants. Further confirmation of the essential role which the exonuclease plays in recombination came from genetic analysis. The T7 exonuclease was shown to be necessary for intragenic and intergenic recombination in several areas of the T7 genetic map; genetic recombination frequencies were found to be decreased from 3 to 18-fold under conditions nonpermissive for the exonuclease. The role of the T7-induced endonuclease (gene 3) in molecular recombination was studied by examining the fate of parental DNA during parent-to-progeny recombination using a shear technique. The T7 endonuclease was found to be necessary for the dispersion of parental DNA in the newly replicated DNA. Concatemers synthesized by either T7⁺ or T7am3 (amber gene 3) phage containing the newly replicated DNA were sheared to the size of mature phage DNA and also to quarter size molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA were interspersed, i.e., the 32P-label from the sheared DNA was found to sediment at the density of recombined DNA. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule was usually found intact, i.e., the ³²P-label from the sheared DNA was found to sediment at the density of hybrid DNA. These results support the previous genetic data (52, 83) that the gene 3 protein is essential for T7 recombination. The role of T7 recombination enzymes in the formation of concatemers was studied by examining selected gene 3 and gene 6 mutants. Results of sucrose gradient analysis showed that DNA concatemers were formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) were absent. Further results showed that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease was eliminated. In a T7am6 infection DNA concatemers formed early were prematurely broken down and accumulated as fragments smaller than mature size phage DNA. In a T7am3am6 (amber in both genes 3 and 6) infection concatemers accumulated and were not matured. These results indicate that concatemers are formed by a process other than normal phage recombination. However, selective defects in the recombination system do interfere with the stability of concatemers. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Life on Earth relies on a set of instructions encoded within an organism’s genome that is passed along from one generation to the next. Inherent to this mechanism of propagation is the need to copy the genetic material before passing it along to the progeny. Errors in this process coupled with stochastic damage will inevitably lead to changes in these instructions and may result in a reduction of fitness or even death of an individual. Yet, these same changes are also responsible for the adaptation mandated by our dynamic environment. Thus, there exists a delicate balance between maintenance and alteration of genetic material that is embodied to a large part at the various intersections of DNA replication, recombination and repair. Homologous recombination (HR) has been well studied and found to play vital roles in many cellular processes from the repair of the harrowing double-stranded break, the restart of a stalled or collapsed replication fork, as well as proper chromosome segregation during meiosis, all with the goal of striking this delicate balance. And yet, while HR is incumbent for the fitness of an organism, if left unchecked this same process can become detrimental by preventing better suited DNA repair pathways, permanently arresting cell cycle progression and creating some of the very problems it was meant to address such as aneuploidy or cancer. Despite a wealth of knowledge, the precise regulatory mechanisms remain an active area of research as they provide likely targets to combat these persistent diseases. Motor proteins that translocate along DNA have been particularly compelling and elusive due to their transitory nature, as well as the inevitability of collisions with bound protein(s) or nucleic acid structures that are likely regulated intermediates in the process. The yeast Srs2 helicase/translocase has long been regarded as the prototypical “anti-recombinase” as it has been shown to dismantle the Rad51 presynaptic filament, but also displays contradictory pro-recombinase functions. In vivo studies of Srs2 have been hampered by its involvement in multiple bioprocesses beyond recombination, while bulk in vitro approaches often produce conflicting results. Recent single molecule imaging of these players has shed light onto their involvement in the regulation of the various stages of the canonical pathway of HR. The Greene laboratory has developed ssDNA curtains to study the pre-synaptic filament and shown that Rad51-ssDNA filaments can create bonafide D-loop intermediates that would be incapable of repair and thus represent a toxic intermediate. These structures persist far longer than the entire process of DSBR in vivo and led us to hypothesize that motor proteins would be a key regulatory element to dismantle improperly paired intermediates for redistribution of the bound proteins and reengagement of the homology search process. Here I extend the use of ssDNA curtains to study Srs2 as it assembles into multimeric complexes to perform long-range disruption of various pre- and post-synaptic filament assemblies that include replication protein A (RPA), Rad51, Rad52, and D-loops. For the first time, direct observation of Srs2 translocating over RPA filaments is provided and shows these proteins are efficiently removed by Srs2. By including Rad52 on the RPA filament, I offer a refined model of the contradictory pro- and anti-recombinase activities of Srs2 through its antagonism of the single-strand annealing pathway in favor of HR. Additionally, Srs2 was found to initiate Rad51 disruption at breaks in the continuity of the filament marked by the persistence of replication protein A (RPA), Rad52, or the presence of an improper D-loop intermediate, the latter of which is efficiently disrupted before continuing translocation. In contrast to the prevailing model, we demonstrate that direct interaction between Srs2 and Rad51 is not necessary for long-range Rad51 clearance. These findings offer insights into the dynamic regulation of crucial HR intermediates by Srs2 and demonstrate that sub-nuclear concentrations of these proteins may be a likely driver for their activities.

Within the life of an organism, its deoxyribonucleic acid (DNA) is constantly bombarded with damaging agents from exogenous and endogenous sources. One of the most deleterious types of damage is the double-stranded break (DSB) in which a continuous strand of DNA is broken in two. As a result, the information stored in their connection is lost. If improperly repaired, a cell will either not survive or transform into a neoplasm. Homologous recombination (HR) is a mechanism by which the cell processes these broken ends and uses proteins called recombinases to search for an undamaged homologous DNA template for repairing the break, the homology search. Generally for eukaryotes, the recombinase, Rad51, performs the homology search. Without it, cells cannot repair spontaneous DSBs by recombination and instead, must use alternative, less efficacious pathways. This type of reparative homologous recombination generally occurs during mitosis and is thus called mitotic recombination. In addition to its role in repair, HR is employed by eukaryotes during the first stage of meiosis to create crossover events, or chiasmata, between DNA homologs. The formation of these chiasmata is necessary for proper segregation of the chromosomes, preventing aneuploidy in the haploid cells destined for sexual reproduction. These crossover events have an added evolutionary benefit of mixing genes between the parental chromosomes, creating allelic diversity in the haploid cells. Eukaryotes have evolved a subset of meioticallyexpressed proteins to mediate this process. Dmc1 is a meiosis-specific, second recombinase that eukaryotes require to properly form these crossover events between homologs. It is not entirely understood why most eukaryotes require a second recombinase specifically designed for meiotic HR. A potential reason for this second recombinase may lie in the preferred templates for recombination that Rad51 and Dmc1 seek. Rad51 is employed mitotically to repair spontaneous DSBs and thus searches for the perfect undamaged copy, the sister chromatid, to prevent the loss of genetic information. Conversely, Dmc1 is employ meiotically to purposely form crossover events between homologs, which carry single-nucleotide polymorphisms (SNPs) between parental chromosomes. Thus, Dmc1 must be able to anneal DNA strands that aren’t perfectly the same. This work uses the single-molecule technique of DNA curtains to understand the factors that effect Rad51 and Dmc1 homologous DNA-capture stability. The first part of Chapter 1 is a historical exploration of homologous recombination research and a review of the current understanding of the pathway. The second part of Chapter 1 discusses human diseases that are associated with the failure to properly repair double-strand breaks. Chapter 2 will explain the single-molecule DNA curtain technique used throughout this work. Chapter 3 will show that Dmc1 is more tolerant of mismatches in captured DNA than Rad51. Chapter 4 will test the limits of Dmc1’s tolerance to imperfect DNA and attempts understand how it accomplishes this tolerance. Chapter 5 will demonstrate that this tolerance of mismatches is mediated by a specific structural element in recombinases, loop L1, and a chimeric Rad51 with a Dmc1-like L1 can tolerate mismatches in vitro and in vivo. Chapter 6 will explore how recombinase mediators such as BARD1 and BRCA1 enhance RAD51’s ability to capture DNA during the homology search.