Characterizing Excluded Strand DNA Interactions with Hexameric Helicases and Determining Roles in Unwinding Mechanisms

DNA replication is an essential process for all living organisms, and errors in this process can lead to genetic mutations and disease. An assembly of protein machinery, termed the replisome, coordinates enzymatic activities at the replication fork. The DNA helicase is the heart of the replisome unwinding double-strand DNA at the head of the progressing replisome and providing single-strand templates for DNA polymerases. Replicative helicases are composed of six subunits, and arranged in a ring-like structure where ATP hydrolysis events provide the energy to translocate upon and unwind the DNA. The mechanism of helicase unwinding has been widely studied, but there are still many aspects that remain unknown. It is generally thought that these helicases encircle one strand of DNA while the other is excluded from the central channel of the helicase. Our lab has previously identified an interaction between the excluded strand and the helicase exterior that was important for unwinding in the archaeal MCM helicase. The steric exclusion model of replicative helicase unwinding was expanded to include the excluded strand interactions in this newly proposed steric exclusion and wrapping (SEW) model. Here, we present work that expands on the SEW model by revealing that the bacterial DnaB and mitochondrial Twinkle replicative helicases also interact with the excluded strand. We have also developed a new single-molecule FRET analysis program to characterize these excluded strand interactions. Although the excluded strand interaction is seen in multiple replicative helicases, we propose distinct roles for the interaction based on functional assays and known differences in replisome architecture across the various organisms. We have also begun to characterize the helicase-excluded strand wrapping interaction in the presence of other replisome components, namely the single-strand binding (SSB) protein. We further characterized the archaeal SSB protein from Sulfolobus solfataricus and provide evidence for a novel DNA-helicase-SSB ternary complex. Overall, this thesis makes significant contributions to the understanding of replicative helicase unwinding mechanisms by expanding upon the current steric exclusion and wrapping model and introduces a novel single-molecule FRET analysis program that we anticipate will be adopted and utilized by others in the field