| This dissertation describes two separate projects. Though the proteins and systems involved in these two studies are very different, in both the use of single-molecule fluorescence techniques has revealed previously unobserved protein-DNA interactions.;The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.;Although prokaryotes lack the machinery utilized by eukaryotes to achieve well organized chromosome segregation, all cells must faithfully segregate their chromosomes in every cell division cycle. In many bacteria, this process is dependent upon a partitioning locus composed of an ATPase called ParA, a DNA binding protein called ParB, and centromere-like binding sites (parS) that are present adjacent to the origin of replication. In Bacillus subtilis, the ParB protein, Spo0J binds to eight parS sites surrounding the origin of replication and spreads along the DNA up to 15 kilobases, forming a nucleoprotein complex. The Spo0J- parS complexes are not only a substrate for Soj/ParB, but they also serve to recruit the highly conserved structural maintenance of chromosomes (SMC) complex to the origin. We have found that Spo0J actually mediates compaction of DNA Using a variety of single-molecule methods, we have observed and characterized the formation of the nucleoprotein complex of purified B. subtilis Spo0J and lambda DNA, both in the presence and absence of a parS site. We propose that Spo0J is capable of forming crosslinks between separate DNA stands. These crosslinks can stabilize loops in the DNA, leading to compaction. |
