DNA replication is one of the most fundamental processes in all living organisms. Its semi-discontinuous nature dictates that the lagging strand is synthesized in short fragments called Okazaki fragments. In eukaryotes, each Okazaki fragment is initiated by an ~ 30-40 nucleotide-long RNA-DNA hybrid primer that is synthesized by Pol α-primase complex. To ensure genomic stability, the RNA primer has to be excised, any misincorporations by Pol α have to be corrected for and finally the resulting nick has to be sealed generating a contiguous strand. This feat is accomplished by a highly coordinated and regulated process called Okazaki fragment maturation. At the center of this process are 5’ nucleases, which are structure-specific nucleases that catalyze the incision of phosphodiester bonds one nucleotide into the 5’ end of ssDNA/dsDNA junctions. Previous structural and biochemical studies have shed some light on the mechanism of FEN1 substrate recognition, its catalysis and regulation. However, many gaps in our understanding of this remarkable nuclease still persist. Moreover, the choice between the short- and long-flap pathways is still elusive. Finally, the mechanism of the coordination among the different enzymatic activities of the polymerase, the nuclease and the ligase during Okazaki fragment maturation is still debatable. In this work, we set out to study FEN1 substrate recognition, catalysis and regulation using single molecule techniques. We show that FEN1 employs a sophisticated substrate recognition mechanism through which it actively distorts the DNA to ~100˚ bent angle. It also displays a remarkable selectivity towards its cognate substrate and avoids off-target substrate by a lock-down mechanism that commits the enzyme for catalysis on cognate substrates while promoting the dissociation of non-cognate substrates. We further characterized FEN1 reaction from substrate binding/bending to product handoff and built a comprehensive kinetic scheme that shows FEN1 releasing its product in two steps. Finally, we uncovered an unprecedented role of FEN1 in the choice between short- and long-flap pathways.

Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites. Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.

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Made available in DSpace on 2016-12-08T17:06:41Z (GMT). No. of bitstreams: 1 murilocopia.pdf: 1751326 bytes, checksum: 9a977527216104faa667914e33ff5fdd (MD5) Previous issue date: 2012-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho consiste em uma etnografia da prática musical das flautas de pífano da banda cabaçal dos Irmãos Aniceto. Tal pesquisa buscou ligar fatos histórico-sociais do grupo em questão e da região do Cariri Cearense com a musicalidade do sujeito da pesquisa. Com tal objetivo realizou-se uma viagem de campo nos primeiros meses de 2011. A metodologia de coleta de dados estabeleceu a relação mestre/aprendiz no intuito de conhecer empiricamente o instrumento musical em questão. Além disso, o aparato teórico sobre etnografia permitiu otimizar o tempo e aprimorar os contatos na viagem que contou com outros sujeitos (que não os Irmãos Aniceto) para formar uma pesquisa contextual e dialógica. Além da relação mestre/discípulo, houve a observação e registro da performance das banda cabaçais da região em eventos como festas de renovação e reisado. Para o entendimento do texto musical e sua relação com o meio, procurou-se identificar padrões de movimentos da relação corpo-instrumento, também conhecidos por padrões acústicos mocionais . O resultado da pesquisa mostrou a forte relação destes padrões com o arcabouço das "categorias nativas" inerentes à sonoridade da banda

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