<p>A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.</p> <p>I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.</p> <p>To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.</p> / Dissertation

<p>Recombination occurs during meiosis to produce new allelic combinations in natural populations, and thus strongly affects evolutionary processes. The model system Drosophila has been crucial for understanding the mechanics underlying recombination and assessing the association between recombination rate and several evolutionary parameters. Drosophila was the first system in which genetic maps were developed using recombination frequencies between genes. Further, Drosophila has been used to determine genetic and environmental conditions that cause variation in recombination rate. Finally, Drosophila has been instrumental in elucidating associations between local recombination rate and nucleotide diversity, divergence and codon bias, as well as helping determine the causes of these associations.</p><p>Here I present a fine-scale map of recombination rates across two major chromosomes in Drosophila persimilis using 181 SNP markers spanning two of five major chromosome arms. Using this map, I report significant fine-scale heterogeneity of local recombination rates. However, I also observed "recombinational neighborhoods", where adjacent intervals had similar recombination rates after excluding regions near the centromere and telomere. I further found significant positive associations of fine-scale recombination rate with repetitive element abundance and a 13-bp sequence motif known to associate with human recombination rates. I noted strong crossover interference extending 5-7 Mb from the initial crossover event. Further, I observed that fine-scale recombination rates in D. persimilis are strongly correlated with those obtained from a comparable study of its sister species, D. pseudoobscura. I documented a significant relationship between recombination rates and intron nucleotide sequence diversity within species, but no relationship between recombination rate and intron divergence between species. These results are consistent with selection models (hitchhiking and background selection) rather than mutagenic recombination models for explaining the relationship of recombination with nucleotide diversity within species. Finally, I found significant correlations between recombination rate and GC content, supporting both GC-biased gene conversion (BGC) models and selection-driven codon bias models. </p><p>Next, I looked at the role of chromosomal inversions in species maintenance by examining the impact of inversions distinguishing species to disrupt recombination rates within inverted regions, at inversion boundaries and throughout the remainder of the genome. By screening nearly 10,000 offspring from females heterozygous for 3 major inversions, I observed recombination rates within an inverted region in hybrids between Drosophila pseudoobscura and D. persimilis to be ~10-4 (similar to rates of exchange for inversion heterozygotes within species). However, despite the apparent potential for exchange, I do not find empirical evidence of ongoing gene exchange within the largest of 3 major inversions in DNA sequence analyses of strains isolated from natural populations. Finally, I observe a strong 'interchromosomal effect' with up to 9-fold higher (>800% different) recombination rates along collinear segments of chromosome 2 in hybrids, revealing a significantly negative association between interchromosomal effect and recombination rate in homokaryotypes, and I show that interspecies nucleotide divergence is lower in regions with larger changes in recombination rates in hybrids, potentially resulting from greater interspecies exchange. This last result suggests an effect of chromosomal inversions on interspecies gene exchange not considered previously.</p><p>Finally, I experimentally tested for a novel male-mediated effect on female recombination rates by crossing males that differed by either induced treatment variation or standing genetic variation to genetically identical females. After assaying recombination frequency in the offspring of these genetic crosses, I fitted these data to a statistical model where I showed no effect of male temperature treatment or male genetic background on offspring recombination rate. However, I did observe a difference of recombination rates of offspring laid 5-8 days post-mating between males treated with Juvenile Hormone relative to control males. Environmental variation in male ability to affect recombination rate in their mates suggests the potential for sexual conflict on optimal proportion of recombinant offspring, perhaps leading to changes in population-level recombination rates with varying levels of sexual selection.</p><p>Overall, my map of fine-scale recombination rates allowed me to confirm findings of broader-scale studies and identify multiple novel features that merit further investigation. Furthermore, I have identified several similarities and differences between inversions segregating within vs. between species in their effects on recombination and divergence, and I have identified possible effects of inversions on interspecies gene exchange that had not been considered previously. Finally, I have provided some evidence that males may impact female recombination rates, although future work should attempt to explore the range of male differences that impact this trait and the mechanism through which males impact the outcome of female meiosis.</p> / Dissertation

The thesis investigates hot carrier relaxation and carrier recombination mechanism of a InN thin film grown on LAO(LiAlO2) substrate with a ultrafast time-resolved photoluminescence apparatus. Carriers were excited with laser pulses of energy 1.5 eV and of pulsewidth 150 fs from a Ti:sapphire laser. The photoexcited carriers relax excessive energy mostly within 10 ps thorough carrier-LO-phonon interaction. The effective carrier-LO-phonon emission times were estimated 197 to 58 fs in the temperature range from 250 to 35 K. The Shockley-Read-Hall coefficient was found around 0.8 ns-1. The Auger recombination was trivial at 35 K and become significant at 250 K. The fitted radiative recombination was much smaller than the theoretical estimate. Both effective carrier-LO-phonon scattering times and the radiative and nonradiative decay rates of the studied m-plane InN were found to be smaller than those of c-plane InN in other reports.

Meiosis, the core engine of sexual reproduction, is a complex process that results in the production of recombinant haploid genomes. In the meioses of Neurospora, worms and mice, gene expression from DNA that lacks a pairing partner is silenced. We posit that this is a two-step process. First, a process called meiotic trans-sensing compares the chromosomes from each parent and identifies significant differences as unpaired DNA. Second, if unpaired DNA is identified, a process called meiotic silencing inhibits expression of genes within the unpaired region and regions sharing sequence identity. Meiotic silencing is mechanistically most likely related to RNAi in other eukaryotes. We used a combination of forward and reverse genetic strategies aimed at understanding the mechanisms of meiotic trans-sensing and meiotic silencing. Here, we present genetic evidence that arguably differentiates the meiotic transsensing step from meiotic silencing, by demonstrating that DNA methylation affects sensing of specific allele-types without interfering with silencing in general. We also determined that DNA sequence is an important parameter scrutinized during meiotic trans-sensing. This, and other observations, led us to hypothesize meiotic recombination as the mechanism for meiotic trans-sensing. However, we find that mutants of key genes required for recombination and chromosome pairing are not required for locus-specific meiotic silencing. We conclude that two interesting possibilities remain: meiotic trans-sensing occurs through a previously uncharacterized recombination pathway or chromosomal regions are carefully compared in the absence of recombination. Finally, forward genetics revealed a novel component of meiotic silencing, Sms-4, encoding the Neurospora ortholog of mammalian mRNP component ELG protein. Unlike previous loss-of-function mutants that abate meiotic silencing by unpaired DNA, Sms-4 is not required for successful meiosis, showing that meiosis and meiotic silencing are distinct, yet overlapping, phenomena. Intriguingly, SMS-4 is the first component to be localized with bulk chromatin in the nucleus, presumably the site of trans-sensing. Finally, we carried out a critical examination of the current evidence in the field and present alternative models for meiotic trans-sensing and meiotic silencing in Neurospora.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 142-144).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 109-140).

Repair of DNA double-strand breaks is critical to the maintenance of genomic stability, and failure to repair these DNA lesions can cause loss of chromosome telomeric regions, complex translocations, or cell death. In humans this can lead to severe developmental abnormalities and cancer. A central pathway for double-strand break repair is homologous recombination (HR), a mechanism that operates during the S and G2 phases of the cell cycle and primarily utilizes the replicated sister chromatid as a template for repair. Most knowledge of HR is derived from work carried out in prokaryotic and eukaryotic model organisms. To probe the HR pathway in human cells, we performed a genome-wide siRNA-based screen; and through this screen, we uncovered cellular functions required for HR and identified proteins that localize to sites of DNA damage. Among positive regulators of HR, we identified networks of pre-mRNA-processing factors and canonical DNA damage response effectors. Within the former, we found RBMX, a heterogeneous nuclear ribonucleoprotein (hnRNP) that associates with the spliceosome, binds RNA, and influences alternative splicing. We found that RBMX is required for cellular resistance to genotoxic stress, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1-dependent manner and through multiple domains, and promotes HR by facilitating proper BRCA2 expression. Screen data also revealed that the mammalian recombinase RAD51 is commonly off-targeted by siRNAs, presenting a cautionary note to those studying HR with RNAi and highlighting the vulnerability of RNAi screens to off-target effects in general. Candidate validation through secondary screening with independent reagents successfully circumvented the effects of off-targeting and set a new standard for reagent redundancy in RNAi screens.

The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.

The foundation of Luecke’s course M: 396 Geometry and Topology is that collaboration amongst mathematicians and biologists caused tremendous gains in DNA research. The field of topology has led to significant strides in understanding of the topological properties of the genetic molecule DNA. Through the integration of biological phenomena and knowledge of topology and Euclidean geometry, biologists can describe and quantize enzyme mechanisms and therefore determine enzyme mechanisms causing the changes. Understanding mathematical applications in contexts outside of mathematics on any level helps to explain why mathematics is a core content area in primary and secondary education. Requiring secondary educators to take such a course could result in mathematics taught with real world application on the secondary level as well as on the graduate level, as shown in Luecke’s course. / text

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy