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==Methods==
==Methods==
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[[Image:|thumb|Figure 1: a) visualization of ssDNA using TIRF microscopy b) Schematic representation of what is visualized in panel a [1] ]]
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[[Image:Direct_Imaging_Figure1ab.jpg‎|thumb|Figure 1: a) visualization of ssDNA using TIRF microscopy b) Schematic representation of what is visualized in panel a [1] ]]
[[Image: Figure4g.jpg|thumb|Figure 4g: Top panel is a schematic of the DNA/bead/trap/SSB used and bottom panel is the visualization of the system using the fluorescent microscope [1] ]]
[[Image: Figure4g.jpg|thumb|Figure 4g: Top panel is a schematic of the DNA/bead/trap/SSB used and bottom panel is the visualization of the system using the fluorescent microscope [1] ]]
In order to visualized ssDNA, researchers tagged the 3’ ends of bacteriophage lambda DNA (48.5 kb) with biotin. Then, they denatured it into single strands before coating the strands with fluorescence tagged single strand binding protein (SSB). These coated ssDNA was then attached  to a glass cover slip covered with streptavidin, a protein which binds strongly to the biotin tag, of a flow chamber. They where then able to visualize the coated strand using total internal reflection fluorescence (TIRF) microscopy (Figure 1a/b, top panel). The tagged SSB was then replaced with wildtype SSB (Figure 1a/b, second panel). The researchers were then able to flow solutions containing fluorescent RecA protein, fluorescein-RecA, through the chamber, over the ssDNA and visualize the binding of RecA to the tethered ssDNA (Figure 1a/b, remaining panels).
In order to visualized ssDNA, researchers tagged the 3’ ends of bacteriophage lambda DNA (48.5 kb) with biotin. Then, they denatured it into single strands before coating the strands with fluorescence tagged single strand binding protein (SSB). These coated ssDNA was then attached  to a glass cover slip covered with streptavidin, a protein which binds strongly to the biotin tag, of a flow chamber. They where then able to visualize the coated strand using total internal reflection fluorescence (TIRF) microscopy (Figure 1a/b, top panel). The tagged SSB was then replaced with wildtype SSB (Figure 1a/b, second panel). The researchers were then able to flow solutions containing fluorescent RecA protein, fluorescein-RecA, through the chamber, over the ssDNA and visualize the binding of RecA to the tethered ssDNA (Figure 1a/b, remaining panels).

Revision as of 11:27, 6 February 2013

“Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA” Jason C. Bell, Jody L. Plank, Christopher C. Dombrowski, and Stephen C. Kowalczykowski

Contents

Background

To ensure their survival, cells have evolved mechanisms by which they both monitor genome integrity and repair damage. The most dangerous type of DNA damage is double stranded breaks (DSB) which can be caused by endogenously generated oxygen radicals, replication forks encountering single stranded DNA breaks or other DNA lesions, or exogenous agents such as ionizing radiation or chemotherapeutic drugs [3]. There are two main options for repairing a DSB: homologous recombination (HR) and non-homologous end joining (NHEJ) [3]. The focus of this paper is proteins, specifically RecA and SSB, involved in homologous recombination in E. coli.

RecA

Diagram of the replacement of SSB protein with RecA [5]
Diagram of the replacement of SSB protein with RecA [5]

RecA is a 38kDA ATPase, a class of enzymes that catalyze the hydrolysis of ATP, found in Escherichia coli that is essential for DNA repair and maintenance [2].

In order to function, the must bind at the site of DNA damage. This occurs in two distinct stages:

  1. Nucleation or the slow aggregation and binding of a few RecA molecules to the DNA lesion.
  2. Growth or the rapid binding of successive RecA proteins in competition with single stranded DNA-binding (SSB) protein to coat the entire lesion.

ATP and ssDNA bind at the RecA-RecA interface cooperatively; together, the complex forms a helical filament which binds dsDNA, searches for homology, and then catalyses strand exchange [2].

Single Strand DNA Binding Protein

In E. coli, the single-stranded DNA-binding protein (SSB) is homotetramer consisting of 18,843 kDa subunits which binds tightly and cooperatively to ssDNA. It is involved in most aspects of DNA manipulation: replication, repair, and recombination. Its functions include prevention of reannealing of ssDNA, protection of ssDNA from nuclease digestion, enhancement of helix destabilization by DNA helicases, primosome assembly, enhancement of replication fidelity [4].

In DNA damage, SSB is involved in induction of the SOS response and recombination repair. During recombination, SSB promotes he binding of RecA protein and strand uptake [4].

Introduction

Methods

Figure 1: a) visualization of ssDNA using TIRF microscopy b) Schematic representation of what is visualized in panel a [1]
Figure 1: a) visualization of ssDNA using TIRF microscopy b) Schematic representation of what is visualized in panel a [1]
Figure 4g: Top panel is a schematic of the DNA/bead/trap/SSB used and bottom panel is the visualization of the system using the fluorescent microscope [1]
Figure 4g: Top panel is a schematic of the DNA/bead/trap/SSB used and bottom panel is the visualization of the system using the fluorescent microscope [1]

In order to visualized ssDNA, researchers tagged the 3’ ends of bacteriophage lambda DNA (48.5 kb) with biotin. Then, they denatured it into single strands before coating the strands with fluorescence tagged single strand binding protein (SSB). These coated ssDNA was then attached to a glass cover slip covered with streptavidin, a protein which binds strongly to the biotin tag, of a flow chamber. They where then able to visualize the coated strand using total internal reflection fluorescence (TIRF) microscopy (Figure 1a/b, top panel). The tagged SSB was then replaced with wildtype SSB (Figure 1a/b, second panel). The researchers were then able to flow solutions containing fluorescent RecA protein, fluorescein-RecA, through the chamber, over the ssDNA and visualize the binding of RecA to the tethered ssDNA (Figure 1a/b, remaining panels).

To visualize RecA filament formation in a more biologically correct system, the researchers generated dsDNA with a ssDNA gap of 8 kilobases with biotin tags on either end. Using microfluidic flow cell in combination with a split beam optical trap and a fluorescent microscope. They bound either end of a single gapped DNA molecule to streptavidin coated beads (Figure 4g, top panel). The optical trap was used to rotate the DNA perpendicular to the flow and to dip the strand into the reaction chamber. The binding of the molecules was measured using the fluorescent microscope (Figure 4g, bottom panel).

Results

References

  1. Bell, Jason C., Plank, Jody L., Dombrowski, Christopher C., and Kowalczykowski. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491, 274 - 278 (2012).
  2. Chen, Z., Yang, H., Pavletich, N.P. Machanism of homolgous recombination from the RecA-ssDNA/dsDNA strucures. Nature 435. 489-494 (2008)
  3. Khanna, K. K. & Jackson, S. P. DNA double-strand breaks: signaling, repair and the cancer connection. Nature Genetics 27, 247 - 254 (2001)
  4. Meyer, R. R. & Laine, P. S. The single-stranded DNA-binding protein of Escherichia coli. Microbiology Review 54, 342-380 (1990)
  5. Cox, Michael M. Motoring along with the bacterial RecA. Nature Reveiws Molecular Cell Biology 8, 127-138 (2007)
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