Construction of Fluorescent Helicase Fusion Proteins for Use in Fret Studies on Helicase Function and Stoichiometry

Abstract

This thesis details the construction of N-terminal fusions of the E. coli helicase DnaB to fluorescent proteins mT-Sapphire and mVenus, and of C-terminal fusions of the hepatitis C virus NS3 helicase (NS3h) to mT-Sapphire and mVenus. These fluorescent fusion proteins were constructed to be used in FRET studies of helicase function and stoichiometry. One example is in assessing the potential oligomerization and cooperativity of NS3h protomers in unwinding RNA. Another potential use is in assessing the stoichiometry of the replisome of E. coli; older studies support a dimeric polymerase model of the replisome. Recent studies however suggest that an active replisome which accommodates three polymerase III cores (the trimeric polymerase complex) may be the active form in vivo and that the previous dimeric polymerase model may have been incorrect. While most evidence supports the dimeric polymerase complex, there are unanswered questions and FRET could potentially answer these questions.