Flap Endonuclease 1 Promotes Telomere Replication and Stability by Distinct Mechanisms on the Leading and Lagging Strands

High fidelity DNA replication is essential for genomic stability and cell survival; this fact is underscored by the redundancy present in DNA replication and repair pathways. The complexity of these pathways is most evident at challenging DNA templates, such as those with repetitive sequence and transcribed loci. Among these challenging templates are telomeres, which are terminal, highly repetitive sequences that maintain genomic stability by preventing aberrant end-to-end chromosome fusions. In the absence of accurate, complete telomere replication, genomic instability results, ultimately leading to cell death or transformation. Here, we describe two unique roles in telomere stability for the DNA replication and repair protein flap endonuclease 1 (FEN1). First, we find that FEN1 maintains telomere stability by facilitating replication fork reinitiation on the lagging strand-replicated telomere. In the absence of FEN1, sister telomere loss (STL) occurs at lagging strand-replicated telomeres. Genetic knockdown-rescue experiments demonstrated that FEN1’s nuclease activity, interactions with DNA repair proteins via its C-terminus, and gap endonuclease activity are essential for preventing STL. Similarly, an analysis of FEN1’s ability to reinitiate stalled replication forks revealed that it is dependent on the same activities as its ability to prevent STL, suggesting that FEN1’s role in reinitiating stalled replication forks is responsible for its ability to suppress STL on the lagging strand. Second, we show that FEN1 maintains telomere stability by limiting telomere fragility on the leading strand-replicated telomere. Strikingly, this activity is biochemically and genetically distinct from FEN’s role in preventing lagging strand- specific STL; FEN1’s ability to suppress telomere fragility depends only on its flap endonuclease activity, while its C-terminal interactions and gap endonuclease activity are dispensable. We show that FEN depletion-induced telomere fragility is increased by RNA polymerase II inhibition and rescued by ectopic ribonuclease H1 expression, suggesting that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap structures that arise following co-directional replisome–RNAP collisions at the telomere. Notably, this is the first known role for FEN1 in leading strand DNA replication, and the first molecular mechanism for telomere fragility at the leading strand. Lastly, we demonstrate that while FEN1 interacts directly with the shelterin protein TRF1, which is required to prevent telomere fragility, this interaction does not contribute to FEN1’s ability to suppress telomere fragility. Together, these data indicate that FEN1 has two functionally separate roles in maintaining telomere replication and stability: preventing STL on the lagging strand by facilitating replication fork reinitiation, and suppressing telomere fragility on the leading strand by processing intermediates that result from replisome–RNAP collisions

MASS SPECTROMETRIC INVESTIGATIONS OF STRUCTURALLY DIAGNOSTIC ION-MOLECULE REACTIONS AND OF MOLECULAR STRUCTURES OF ASPHALTENES

Tandem mass spectrometry (MS/MS) has proven to be a powerful tool for the molecular-level characterization of components of complex mixtures due to its ability to study ionized molecules with high sensitivity, selectivity and specificity. By combining MS/MS with liquid chromatography (LC) to enable front end separation of complex mixtures, trace level analysis of components in these mixtures is possible. Hence, LC-MS/MS has proven invaluable in the separation and characterization of saturated, alkyl aromatic, and polycyclic aromatic hydrocarbon constituents of crude oil. The most common MS/MS method involves collision-activated dissociation of mass-selected ions. Additionally, MS/MS strategies utilizing functional-group or isomer specific ion-molecule reactions have been recognized as a fast and efficient way to identify specific analytes among mixture components. However, the mechanisms of many gas-phase reactions, including dissociation and ion-molecule reactions, are not easily deciphered using MS alone. Thus, density functional theory (DFT) calculations may be used in combination with MS/MS data to derive reaction pathways leading to the formation of intermediate and product ions. Knowledge of the structures of these intermediate and product ions and pathways to their formation may be used to develop new mass spectrometry methodologies for elucidation of structures of unknown components in mixtures and to identify better reagents for functional-group and isomer specific ion-molecule reactions

Protein identification and characterization through peptide mass spectrometry

The illicit usage of toxic substances is increasing rapidly around the world, creating a need for comprehensive chemical methods for detecting and quantitating toxic agents that might be used in criminal activities or terror attacks. There are several toxic proteins that could potentially be used in bioterrorism or biocrime. For example, the plant toxins ricin and abrin are readily available, very toxic, and easily produced, and are therefore frequently involved in biocrime incidents. Another threat agent is the extremely poisonous botulinum neurotoxin, which is among the most toxic substances known. Forensic analyses of samples potentially containing these diverse and very toxic agents therefore require analytical methods capable of detecting trace amounts of the target analyte in complex mixtures. This thesis describes the development of new methods for peptide mass spectrometry that offer improved performance in forensic toxin analysis. A galactose affinity method was developed for the enrichment and subsequent analysis of ricin, abrin and botulinum neurotoxin. The method’s applicability was confirmed during a forensic investigation into illegal toxin preparations. Additionally, the investigations generated new information on the structural properties of ricin that will facilitate the forensic matching of samples to sources. Details of botulinum neurotoxin’s sophisticated intoxication mechanisms were determined by using a broad analytical approach to study the importance and heterogeneity of SV2C glycosylation. Overall, the results and procedures presented in this thesis will help to improve analytical capabilities relevant to the detection and prevention of biocrime and bioterrorism. More generally, it provides methodological guidance and useful strategies for researchers in peptide mass spectrometry

Effect of yoghurt processing on beta-casomorphin 5 and beta-casomorphin 7 concentrations using novel liquid chromatography-mass spectrometry methods

The effect of yoghurt processing on the concentration of beta-casomorphin 5 and beta-casomorphin 7 was investigated. Liquid chromatography-mass spectrometry methods were developed and validated. The milk beta-casomorphin 7 range was 0.13 and 2.38 ng/g and beta-casomorphin 5 was below limit of detection. Beta-casomorphin 7 was completely degraded when milk was fermented with individual bacteria or a mixture of L. delbrueckii ssp. bulgaricus and S. thermophilus at pH 4.5 and after 7 days of yoghurt storage

Structure-function relationships of variegin: A novel class of thrombin inhibitors

Ph.DDOCTOR OF PHILOSOPH