%0 Generic
%9 Doctoral Dissertation
%A Graham, Brian
%D 2014
%F pittir:22749
%K archaea helicase SSB primase primosome DNA replication archaeal MCM DnaG Sulfolobus solfataricus Sso SsoMCM SsoDnaG SsoSSB
%T Mechanistic and Functional Characterization of Components of the Sulfobolus solfataricus Primosome
%U http://d-scholarship-dev.library.pitt.edu/22749/
%X DNA replication is a complex process that involves an organism’s ability to faithfully replicate and maintain its genomic code. One of the initial stages of DNA replication is the separation of double-stranded DNA into complementary single-stranded DNA. This action is performed minimally by the replicative DNA helicase. DNA helicases are present in all three domains of life and are essential for cellular survival. Mutations to helicases can lead to a predisposition for susceptibility to cancers and other diseases. The actual mechanism for DNA unwinding and processing at the replication fork, however is unknown. We chose a model archaeal system (Sulfolobus solfataricus - Sso) that is homologous to eukaryotes, but is simplified in its DNA replication machinery. The DNA replicative helicase in Sso is the homohexameric minichromosome maintenance protein (MCM) which is homologous to the eukaryotic heterohexamer Mcm2-7. I have discovered the novel mechanism for unwinding in Sso which we named the steric exclusion and wrapping model (SEW). The SEW model involves MCM encircling the DNA leading strand and sterically excluding the lagging strand which then physically wraps around the exterior of the helicase. We hypothesize this wrapping protects the single-stranded DNA from degradation to allow for elongation on the lagging strand. The binding path on the exterior of the helicase was further characterized through the mutation of two basic residues that we determined electrostatically interact with the single-stranded DNA. Additionally, I have characterized a conserved single point mutation that disrupts hexamerization, implying additional functional significance for the motif in which it is located. Finally, I characterized interactions within the archaeal primosome including the atypical physical interaction between eukaryotic-like helicase MCM and bacterial-like primase DnaG and the functional relationship between MCM and single-stranded binding protein. My thesis provide a significant contribution to the overall understanding of the mechanism and function of proteins in the archaeal primosome, which will be applicable to more complex eukaryotic systems.