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Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate

Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate

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GALAL, Wiebke, 2012. Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate

@phdthesis{Galal2012Worki-20675, title={Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate}, year={2012}, author={Galal, Wiebke}, address={Konstanz}, school={Universität Konstanz} }

Galal, Wiebke eng deposit-license Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate Galal, Wiebke 2012-10-15T08:53:21Z DNA replication is an essential process whereby an entire double-stranded DNA is copied to produce a second, identical DNA double helix. This process requires the concerted action of a large number of proteins. The basic mechanism governing this vital macromolecular event is conserved among prokaryotes, archaea and eukaryotes. The helicase unwinds the DNA double helix into two individual strands. Single strand binding proteins coat the single-stranded DNA to prevent the strands from reannealing. Primase is a polymerase that synthesizes the short primers needed to start the replication process. Primases are necessary because DNA polymerases can only extend a nucleotide chain, not start one. The DNA polymerase starts at the 3’ end of the primer, and, using the original strand as a template, begins to synthesize a new complementary DNA strand by linking the 5’ phosphate group of an incoming nucleotide to the 3’ hydroxyl group at the end of the growing nucleic acid chain.<br /><br />The studies presented in this thesis are described in three recently published papers. In the publication titled “ Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase ε in rolling circle DNA synthesis”, we describe the purification and biochemical characterization of the human replicative DNA helicase, the CMG complex, which consists of Cdc45, Mcm2-7 and GINS. Another manuscript (“Characterization of the DNA primase complex isolated from the archaea, Thermococcus kodakaraensis”) focuses on the archaeal DNA primase, which consists of a small catalytically active subunit and large regulatory subunit devoid of enzymatic activity. While this structure is similar to the eukaryotic DNA primase, we found that the archaeal complex initiates DNA as well as RNA chains de novo, while the eukaryotic complex only initiates RNA chains. Finally, we studied the biochemical properties of a previously uncharacterized nuclease that localizes to the replication components (“A novel DNA nuclease is stimulated by association with the GINS complex”).<br /><br />Classically, the enzymes involved in replication have been characterized under conditions in which each protein is examined in the absence of other replication components. Rolling circle assays have been recently developed for prokaryotic systems to study the jointly action of replication proteins during DNA synthesis. The primase and the CMG studies presented in this thesis describe the development of an in vitro rolling circle method used to examine leading and lagging strand synthesis with both archaeal and eukaryotic proteins. Our detailed studies in the eukaryotic system revealed that the putative leading strand polymerase ε, in concerted action with the helicase, supported the synthesis of long DNA chains while DNA polymerase δ, the putative polymerase involved in lagging strand synthesis, did not support the synthesis of long DNA chains.<br /><br />Overall, the availability of rolling circle assays will be of great advantage in analyzing the functions played by the various components of the DNA replication machinery of human cells.<br /> 2012-10-15T08:53:21Z 2012

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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