Structural and Biophysical Studies of Single-Stranded DNA Binding Proteins and dnaB Helicases, Proteins Involved in DNA Replication and Repair
The processes of DNA replication, repair and recombination have been studied for many years using model systems such as E. coli. One key aspect of DNA replication is the role of proteins at the replication fork. It is important to understand the interactions of these proteins with the other proteins involved in the process or with a DNA substrate. In PART I of this dissertation, studies of the Archaeal Aeropyrum pernix and Archaeoglobus fulgidus single-stranded DNA binding protein are presented. Archaea are more closely related to eukaryotes than the commonly studied E. coli model system. The SSB proteins have an involvement in Okazaki fragment processing. The FEN-1 enzyme removes DNA flaps that are 3 to 5 nucleotides long. If the length of the flap is more than 5 to 7 nucleotides, two other proteins are then involved in the removal of these flaps, the SSB protein that binds to the flap, and dna2 needed to shorten the flap. Using PCR and molecular cloning techniques and protein chemistry, milligram quantities of the purified Ape SSB protein were obtained. Preliminary attempts to crystallize the full length protein failed, most likely due to highly flexible C-terminal region. To overcome this problem, a truncated version of the protein was prepared. The truncated protein crystallized in two different crystallization conditions. One produced beautiful large 1mm hexagonal crystals that appeared twinned on diffraction. The other crystal form, rod shaped small crystals, diffracted to almost 1.6 Å. The structure of the truncated protein was solved using molecular replacement with an already known SSB protein from the species Sulfolobus sulfataricus. Biophysical studies, Dynamic Light Scattering (DLS) and Fluorescence Anisotropy, have been carried out to characterize the Ape SSB protein. In PART II, work done on DNA helicases (dnaB) from different organisms is presented. Expression and purification protocols for Vibrio cholerae dnaB were successfully established and crystal screens set up. Few hits were obtained in the screens but expansion of some of the conditions did not yield any crystals. Cloning, expression and purification protocols were established for Yersinia pestis dnaB and Helicobacter pylori dnaB.
School:University of Toledo
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:ssb proteins single stranded dna binding archaeal okazaki fragment processing structure of helicases
Date of Publication:01/01/2007