Date of Award
Doctor of Philosophy (PhD)
Chemistry & Biochemistry
Charles S. McHenry
During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and trigger release from the lagging strand. A hypothesis in the literature proposes that the τ subunit (of the DnaX complex) directly senses completion of an Okazaki fragment. An alternative model suggests that the polymerase subunit senses conversion of a gap to a nick. I show using a novel phenyldiazirine photo-crosslinker linked to the 5-position of thymidylate that the τ subunit is not in position to distinguish gapped DNA from nicked DNA. The α subunit (the polymerase) is positioned to serve as the processivity sensor. Upon encountering duplex DNA, the polymerase likely changes conformation triggering its release from the lagging strand and the β processivity clamp, modulating its own affinity. Unrepaired replication forks dissociate from the helicase and suffer collapse. PriA recognizes stalled replication forks and initiates interactions to reload the helicase and activate a previously stalled fork. I used a FRET helicase assay to develop a PriA- dependent helicase loading system in E. coli and B. subtilis and to identify a minimal substrate to support a photo-crosslinking study also discussed here. I discovered that PriA's ATPase activity dictates substrate specificity. I also show that PriA serves as a checkpoint protein by blocking the replicase from binding to stalled replication forks distinguishing between an alternative model. SPP1 is a bacteriophage that infects B. subtilis. It encodes its own initiation proteins (origin binding protein, primosomal proteins, helicase, and single-strand binding protein (SSB)) but requires its host's primase and major replicative polymerase to replicate its genome. Both host and phage SSBs can support a reconstituted SPP1 system, but phage SSB does not support a reconstituted B. subtilis system. Using the B. subtilis FRET helicase assay, I show that phage SSB can substitute for the host's SSB in helicase reloading. Therefore the defect in the reconstituted system is not at the level of helicase loading or function and must occur after the helicase is loaded. I also show an absolute requirement on all SPP1 components in helicase reloading, including the origin binding protein (in a non-origin-containing template), which suggests a new role for this protein. In collaboration with Tim Lohman's lab at Washington University, I have contributed to a study into the functions of the C-terminal tails of SSB. SSB functions as a homotetramer whose four C-terminal tails interact with many other proteins necessary for DNA replication and repair. In an in vivo assay, an SSB variant that has two functional C-terminal tails supports viability in E. coli. An SSB variant that has one C- terminal tail is dominant lethal. In a reconstituted rolling circle E. coli replication system, there is a defect in coupled synthesis that causes a two-fold decrease in lagging strand synthesis relative to the leading strand using the variant with one C-terminal tail. This is significant, but does not sufficiently account for the lethality in vivo. Using the E. coli FRET helicase assay, I show that the variant with one C-terminal tail causes the PriA replication restart pathway to be inactive. Presumably, all replication forks suffer a collapse if leading and lagging strand synthesis are uncoupled. Thus, the replication restart pathway becomes even more critical. The SSB variant with one C-terminal tail does not support this pathway, which provides an explanation for the lethality. Photo-crosslinking is used to probe the dynamics of E. coli primosomal proteins on a replication fork in the replication restart pathway. Both the specific locations on the DNA where PriA and the other protein machinery are bound and how the proteins change position as the complex protein machinery is assembled are identified. I have determined the binding positions of SSB and revealed a novel interaction between a replication fork and SSB. I have also determined that PriA excludes SSB at the replication fork. When the DnaB helicase is loaded onto the lagging strand, it interacts with the displaced strand and diffuses at least three nucleotides into the duplex. Interestingly, DnaB is seen to only make weak contact with the lagging strand arm.
Manhart, Carol Michelle, "Investigating protein-DNA interactions at replication forks by photo-crosslinking" (2013). Chemistry & Biochemistry Graduate Theses & Dissertations. 78.