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|Title:||Structural Studies of DNA Replication Machinery in Archaea|
|Author(s):||Bae, Brian Bongik|
|Doctoral Committee Chair(s):||Nair, Satish K.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||DNA replication is a fundamental process present in all living organisms. It is the means by which organisms transfer their genetic information to their offspring to ensure the propagation of their lineage. One of the essential features of DNA replication is to separate the DNA double helix to generate a replication fork by exposing two single strands that serve as templates. Studies on the replication fork have identified large number of proteins involved in this process. The underlying mechanisms of DNA replication has been intensively studied in both bacteria and eukaryotes. However, DNA replication in archaea, the third domain of life, is not well characterized. Accumulating evidence indicates that archaeal DNA replication machinery is more homologous to that of eukaryotes but presents a simpler molecular architecture. The aim of my thesis work is to use biochemical and biophysical methods to investigate the molecular basis of structure-function relationship, protein-protein interaction, and protein-DNA interaction in the archaeal DNA replication machinery.
The separation of the two strands of the DNA double helix is mediated by the ATP dependent replicative DNA helicase, Mini Chromosomal Maintenance (MCM) protein. Although the MCM proteins have been biochemically characterized, limited structural information precludes further understanding of their mode of action. To address this limitation, we have determined the crystal structure of an MCM homolog from archaea Methanopyrus kandleri at 1.9 A resolution. The monomeric structure reveals unique insertions and motifs, necessary for its helicase action, which place the MCM helicases as a distinct clade of AAA+ (ATPases Associated with various cellular Activities) ATPase. We reconstitute the functional hexameric form by superimposing the monomer structure onto a cryo EM reconstruction of an MCM hexamer. The reconstructed model provides insight into the molecular basis of inter-subunit interaction implicated in the ATP-dependent helicase function.
The single stranded DNA generated at the replication fork by the actions of the MCM helicase is not stable and susceptible to hydrolysis. The naked DNA is protected by an accessory protein, the single stranded DNA (ssDNA) binding protein called Replication Protein A or RPA. In order to elucidate the structure of archaeal RPA, we identify a core scaffold that retains complete biological function and harbors all of the structural motifs common amongst archaeal RPAs. We have determined the 2.4 A crystal structure of this core scaffold (RPA57-411), as well as that of the tandem OB fold (57-260) and zinc binding motif (268-411). The structures reveal a linear arrangement of three OB (oligonucleotide/oligosaccharide binding) folds. The zinc-binding motif is inserted in the third OB fold, which is involved in subunit dimerization rather than ssDNA binding. We biochemically and biophysically characterize ssDNA binding activity to elucidate a model for ssDNA binding by the RPA dimer. These structural and biochemical studies reveal novel domain architecture, and provide insights regarding molecular basis of interaction.
Synthesis of the daughter DNA strands is catalyzed by DNA polymerases at the replication fork. Error-prone DNA polymerases can bypass abnormal DNA templates, which are persistent in cells due to various endogenous and environmental DNA damaging agents. Although these error-prone DNA polymerases can rescue a stalled replication fork, they inevitably introduce point or frame-shift mutations, eventually compromising the genomic integrity. We aimed to study how the error-prone polymerase is recruited to replication fork. We hypothesize that the polymerase is recruited through the direct interaction with Proliferating Cell Nuclear Antigen (PCNA), the trimeric, ring shaped processivity factor of the replicative polymerase. We determined the crystal structure of PCNA interacting domain of error-prone DNA polymerase complexed to PCNA from archaea Methanosarcina acetivorans. The co-crystal structure revealed that determinant of polymerase-PCNA interaction extends beyond those of the PCNA-binding peptide that typically characterize such PCNA-protein complexes.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
|Date Available in IDEALS:||2014-12-17|