Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerases carry out these DNA transactions by passing one segment of DNA through the other by using a reversible, enzymebridged double strand break. The transient enzyme/DNA adduct is mediated by a phosphodiester bond between the active-site tyrosine and a backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this cleavage/religation. We designed a unique oligonucleotide substrate with a pair of fluorophores straddling the topoisomerase II cleavage site, allowing the use of FRET to monitor the opening of the DNA gate. The DNA substrate undergoes an enzyme-mediated transition between a closed and open state in the presence of ATP, similar to the overall topoisomerase II catalyzed reaction. Single-molecule fluorescence microscopy measurements demonstrate that the transition has comparable rate constants for both the opening and closing reaction during steady-state ATP hydrolysis, with an apparent equilibrium constant near unity. In the presence of AMPPNP, a reduction in FRET occurs, suggesting an opening or partial opening of the DNA gate. However, the single-molecule experiments indicate that the open and closed states do not interconvert at a measurable rate.fluorescence microscopy ͉ single-molecule kinetics ͉ FRET ͉ enzyme dynamics T opological entanglements, including supercoiling, knotting, and catenation, can arise during various processes involving DNA and are removed by DNA topoisomerases (1-3). These complex tasks of regulating chromosome structure are achieved with the simple but elegant chemistry of transesterification reactions. The active-site tyrosine of topoisomerase carries out a nucleophilic attack to form a covalent adduct with a DNA backbone phosphate, thus generating a transient, reversible break to enable topological transformations in DNA. The reversal of this transesterification reaction reforms the DNA backbone bond and restores the enzyme tyrosyl residue. Type II topoisomerases are dimeric in structure and generate a transient double strand break, hence permitting another DNA segment to pass through this enzyme-bridged DNA gate. During the catalytic cycle, a DNA segment has to traverse the entire symmetry axis of the dimer interface, including the enzyme-mediated DNA break serving as a DNA gate during strand passage (4). The reactions at the DNA gate thus involve DNA cleavage, opening and closing of the gate, and religation of the DNA. To accommodate this large-scale DNA movement, the enzyme has to undergo a series of concerted conformational changes that are fueled in part by ATP binding and hydrolysis.Biochemical analysis has demonstrated that ATP can trigger the capture of a DNA segment by the N-terminal clamp and promotes the passage of this DNA segment through the transient DNA break (5, 6). This reversi...

DNA replication of the mitochondrial genome is unique in that replication is not primed by RNA derived from dedicated primases, but instead by extension of processed RNA transcripts laid down by the mitochondrial RNA polymerase. Thus, the RNA polymerase serves not only to generate the transcripts but also the primers needed for mitochondrial DNA replication. The interface between this transcription and DNA replication is not well understood but must be highly regulated and coordinated to carry out both mitochondrial DNA replication and transcription. This review focuses on the extension of RNA primers for DNA replication by the replication machinery and summarizes the current models of DNA replication in mitochondria as well as the proteins involved in mitochondrial DNA replication, namely, the DNA polymerase γ and its accessory subunit, the mitochondrial DNA helicase, the single-stranded DNA binding protein, toposiomerase I and IIIα and RNaseH1.

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