Ribonucleotides, also known as ribonucleoside monophosphates (rNMPs), are the most abundant non-canonical nucleotides incorporated into genomic DNA. Despite the relevance, information about their repair pathways, consequences, and profiles is still lacking. Exploiting the use of oligonucleotides containing rNMPs in a molecular approach to generate various RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in cells, we show that mispaired rNMPs embedded into genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in E. coli and S. cerevisiae cells. In addition, we discovered that paired rNMPs in DNA are targets of both RNase H type 2 and nucleotide excision repair (NER) in yeast. Also, we report atomic force microscopy (AFM)-based single molecule elasticity measurement, molecular dynamics simulation, and nuclear magnetic resonance spectroscopy results, showing that rNMPs in short DNA duplexes can change the elastic and structural properties of DNA. Lastly, we developed ribose-seq, a method for capturing rNMPs embedded in DNA. High-throughput sequencing of rNMP-captured molecules from the yeast S. cerevisiae revealed widespread but non-random rNMP distribution with preferences in base composition of rNMPs and neighboring DNA sequence context in both nuclear and mitochondrial DNA. With ribose-seq, systematic profiling of rNMP incorporation into genomic DNA is achieved, potentially allowing determination of specific signatures of rNMPs in DNA which could help to better understand the nature of rNMP repair mechanisms, effect of rNMPs on DNA mechanical properties and structure, and eventually rNMP impact on genome integrity.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

Human immunodeficiency virus type 1 (HIV-1) has caused more than 70 million infections worldwide since its discovery, with half of the infected already died by the end of 2011 as a result of HIV-progression to acquired immunodeficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) is capable to extend the lifespan of HIV patients but economic burden and emergence of resistant HIV strains pose immediate problems in the care of HIV patients. Furthermore, HAART cannot clear virus. Therefore, tremendous efforts have tried to develop an effective HIV vaccine in the last thirty years but only partial efficacy (31%) was achieved in the recent Thai RV144 clinical trial. Hence, new vaccine and understanding the mechanism are required now and in the future. In this study, two novel DNA vaccine strategies that utilized programmed death-1 (PD-1) or its isoform to improve immunogenicity of DNA vaccine for HIV-1 Gag p24 by acting on dendritic cells are described. The molecule PD-1 delivers negative regulatory signals to T cells through interacting with its ligands PD-L1 and PD-L2, while blocking this signal could functionally rescue the “exhausted” T cells during chronic infection such as HIV-1. The first DNA vaccine involves the fusion between HIV-1 Gag p24 antigen and soluble PD1 for effective targeting to DCs while improving antigen uptake and DC maturation, which in turn elicited consistently high frequencies of HIV-1 Gag-specific, broadly reactive, polyfunctional, long-lived and cytotoxic CD8+ T cells, in addition to robust anti-Gag antibody titers in mouse. The mechanism behind the action of this vaccine (sPD1-p24fc) is based on engagement of cross-presentation to CD8+ T cells, and induction of Th1 cytokines. The second DNA vaccine utilized a novel isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain discovered in healthy PBMC donors. Interestingly, Δ42PD1 does not engage PD-L1/PD-L2 but its recombinant form could induce pro-inflammatory cytokines. When Δ42PD1 was used as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen (sΔ42PD1-p24fc), enhanced DC uptake was also observed. When mice was vaccinated with this vaccine, significantly enhanced anti-p24 IgG1/IgG2a antibody, p24-specific T cells responses with functionally improved proliferative and cytolytic capacities were also identified. Importantly, both of these vaccines enhanced antigen-specific immunity and provided protection against pathogenic viral challenge as well as tumor growth in mice. Overall, the induction of high frequency of durable and protective Gag-specific T cell immunity, especially CD8+ T cell immunity using these two vaccines have important implications for vaccine development and immunotherapy against HIV-1 and other pathogens. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

DNA sequencing is the process of determining the identities of the nucleotides that make up a molecule of DNA. The rapid pace of advancements in sequencing technologies in recent years have made it possible to simultaneously determine the sequences of hundreds of millions of short DNA fragments. The ability to perform sequencing with such high throughput has revolutionized the study of biological systems, but the types of questions that can be answered through sequencing-based experiments can be limited by the presence of different kinds of noise and biases in these experiments. One class of applications of high-throughput sequencing involves identifying genetic variation, such as finding rare mutations in the genomes of cancerous cells. In these applications, the sensitivity with which rare genetic variants can be detected is limited by the relatively high rate with which current DNA sequencing technologies incorrectly identify nucleotides. In the first half of this thesis, we present a method for dramatically reducing the rate at which these incorrect identifications occur. Our method, called circle sequencing, creates redundant copies of the sequence of each input molecule of DNA. This is accomplished by circularizing each DNA fragment and performing rolling circle amplification on these circles with a strand-displacing polymerase. The resulting products consist of several physically linked copies of the original sequence in each fragment. When these products are sequenced, this informational redundancy protects against random errors introduced during sequencing, allowing for highly accurate recovery of the original sequence of each input molecule. By eliminating the vast majority of incorrectly identified nucleotides from the resulting data, our method enables the sensitive detection of rare variants and opens up exciting new questions involving such variants to direct measurement by sequencing. An entirely different application of high-throughput sequencing is to selectively capture and sequence stretches of DNA or RNA that are participating in a process of interest within a cell. The accuracy of quantitative inferences made by this type of experiment can be severely impacted, however, by biases introduced during the experimental manipulations used to isolate biologically relevant fragments of DNA from cells. Ribosome profiling is an experimental technique that consists of sequencing short stretches of messenger RNAs that are protected from nuclease digestion by the presence of a bound ribosome. The resulting data represents millions of snapshots of the locations of actively translating ribosomes. In theory, these snapshots can be used to determine how long ribosomes take to translate each type of codon by quantifying how often ribosomes are observed positioned over that codon. In practice, different studies in yeast attempting to do this have reached contradictory and counterintuitive conclusions. In the second half of this thesis, we perform a large-scale comparative analysis of data from many different ribosome profiling experiments in order to resolve these contradictions. We identify a previously unappreciated source of systematic bias in a subset of these experiments. This bias prevents these experiments from accurately measuring ribosomes in proportion to how long they spend at each position in vivo. Understanding this bias provides insight into the true signatures of translation dynamics in yeast and offers important guidance for the future design and interpretation of sequencing-based approaches to measuring these dynamics.

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