Nathan A. Tanner

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.

E. coli DNA replication in the absence of free β clamps

During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging‐strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging‐strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging‐strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single‐molecule experiments, we show that replication complexes pre‐assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA‐primer synthesis. These results broaden our understanding of lagging‐strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps.

Visualizing DNA Replication at the Single-Molecule Level

Recent advances in single-molecule methodology have made it possible to study the dynamic behavior of individual enzymes and their interactions with other proteins in multiprotein complexes. Here, we describe newly developed methods to study the coordination of DNA unwinding, priming, and synthesis at the DNA-replication fork. The length of individual DNA molecules is used to measure the activity of single replisomes engaged in coordinated DNA replication. First, a tethered-particle technique is used to visualize the formation and release of replication loops. Second, a fluorescence imaging method provides a direct readout of replication rates and processivities from individual replisomes. The ability to directly observe transient reaction intermediates and characterize heterogeneous behavior makes these single-molecule approaches important new additions to the tools available to study DNA replication.

Single-Molecule Observation of Prokaryotic DNA Replication

Recent advances in optical imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and study the dynamic properties of processes that are challenging to elucidate using ensemble-averaging techniques. The use of single-molecule approaches has proven to be particularly successful in the study of the dynamic interactions between the components at the replication fork. In this section, we describe the methods necessary for in vitro single-molecule studies ofprokaryotic replication systems. Through these experiments, accurate information can be obtained on the rates and processivities of DNA unwinding and polymerization. The ability to monitor in real time the progress of a single replication fork allows for the detection of short-lived, intermediate states that would be difficult to visualize in bulk-phase assays.

Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

A single molecule DNA flow stretching microscope for undergraduates

The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no special vibration isolation and is capable of measuring DNA replication at the single molecule level. We discuss two laboratory experiments suitable for advanced undergraduates using our microscope.

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.

METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B

Recent advances in single-molecule methodology have made it possible to study the dynamic behavior of individual enzymes and their interactions with other proteins in multiprotein complexes. Here, we describe newly developed methods to study the coordination of DNA unwinding, priming, and synthesis at the DNA-replication fork. The length of individual DNA molecules is used to measure the activity of single replisomes engaged in coordinated DNA replication. First, a tethered-particle technique is used to visualize the formation and release of replication loops. Second, a fluorescence imaging method provides a direct readout of replication rates and processivities from individual replisomes. The ability to directly observe transient reaction intermediates and characterize heterogeneous behavior makes these single-molecule approaches important new additions to the tools available to study DNA replication.

VISUALIZING DNA REPLICATION AT THE SINGLE-MOLECULE LEVEL

Recent advances in single-molecule methodology have made it possible to study the dynamic behavior of individual enzymes and their interactions with other proteins in multiprotein complexes. Here, we describe newly developed methods to study the coordination of DNA unwinding, priming, and synthesis at the DNA-replication fork. The length of individual DNA molecules is used to measure the activity of single replisomes engaged in coordinated DNA replication. First, a tethered-particle technique is used to visualize the formation and release of replication loops. Second, a fluorescence imaging method provides a direct readout of replication rates and processivities from individual replisomes. The ability to directly observe transient reaction intermediates and characterize heterogeneous behavior makes these single-molecule approaches important new additions to the tools available to study DNA replication.