Gene machines

Abstract:

Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFRET assays utilizing doubly labeled variants of Escherichia coli DNA polymerase I were pivotal in identifying and characterizing a partially closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labeling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analyzing the conformations of DNA polymerases in vitro and in vivo.

Abstract:

The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that are preferentially localized to the replication origin region (ori), close to the long axis of the cell. A replication-dependent increase in the fraction of immobile molecules, together with a proposed catalytic cycle of ~1.8 s, is consistent with the majority of active TopoIV molecules catalyzing decatenation, with a minority maintaining steady-state DNA supercoiling. Finally, we show that the MukB-ParC interaction is crucial for timely decatenation and segregation of newly replicated ori DNA.

Abstract:

To achieve single-molecule sensitivity and thus have the ability to detect single diffusing fluorophores, careful alignment of the microsecond-alternating laser excitation (μs-Alex) setup is crucial. The following protocol describes routine alignment for 2c-ALEX (532 nm/635 nm) with spectral windows G(550-620)R(650-750).

Abstract:

Single-molecule fluorescence resonance energy transfer (smFRET) has been widely applied to the study of fluorescently labeled biomolecules on surfaces and in solution. Sorting single molecules based on fluorescent dye stoichiometry provides one with further layers of information and also enables "filtering" of unwanted molecules from the analysis. We accomplish this sorting by using alternating laser excitation (ALEX) in combination with smFRET measurements; here we describe the implementation of these methodologies for the study of biomolecules in solution.

Abstract:

This protocol describes the construction of a microsecond-alternating laser excitation (μs-ALEX) using two lasers, a green 532-nm acousto-optically modulated laser and a red 635-nm directly modulated laser.