Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

Biochemical Characterization of Human Exonuclease 1 Protein-protein Interactions

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.

Enzymatic Polymerization of High Molecular Weight DNA

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.