Use of Chromophoric Ligands to Visually Screen Co‐Crystals of Putative Protein‐Nucleic Acid Complexes

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Distinguishing between crystals of protein?nucleic acid complexes and those containing protein alone is a common problem in structural studies of protein?nucleic acid interactions. Currently, there are several methods available for detecting nucleic acid in crystals, including gel electrophoresis, SYBR Gold fluorescence dye staining, and methyl violet staining. However, they require either that the crystals be sacrificed or access to a fluorescence microscope. In this protocol, we describe an approach that allows direct visualization of either the presence or absence of oligonucleotides in crystals grown from solutions containing both protein and nucleic acid?labeling with the Cy5 dye. In addition to offering the advantage of being able to distinguish between crystals of complex and protein alone with the naked eye or a light microscope, crystals of covalently Cy5?labeled DNA can be directly used for X?ray diffraction data collection. Curr. Protoc. Nucleic Acid Chem. 46:7.15.1?7.15.8. © 2011 by John Wiley & Sons, Inc.

Keywords: crystallization; oligonucleotide conjugates; protein?nucleic acid interactions; X?ray crystallography

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Introduction
Basic Protocol 1: Co‐Crystallization of a Duplex Composed of 5′‐Cy5‐Labeled DNA Primer and Unlabeled DNA Template with DNA Polymerase Eta from Saccharomyces cerevisiae (scPol η)
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Co‐Crystallization of a Duplex Composed of 5′‐Cy5‐Labeled DNA Primer and Unlabeled DNA Template with DNA Polymerase Eta from Saccharomyces cerevisiae (scPol η)

Materials

Purified and desalted 5′‐Cy5‐labeled primer strand (5′‐Cy5‐GTGGTCAAG‐3′; Integrated DNA Technologies; http://www.idtdna.com/Home/Home.aspx)
Purified and desalted native DNA template strand 5′‐CTTCTTGACCAC‐3′ (IDT)
sc Pol η enzyme, catalytic core, residues 1‐513 (sc Pol η_1‐513 ) (Trincao et al., ; Alt et al., )
Ice
Magnesium chloride (Fisher Scientific)
2′‐Deoxyadenosine‐5′‐[(α,β)‐methyleno]triphosphate (dAMPcPP; Jena Bioscience)
PEG Suite crystallization screen (Qiagen) containing reservoir solution

Microcentrifuge tubes
Various pipetman pipets and tips (Gilson)
Benchtop centrifuge
Heating block to anneal the primer and template strands
Vortex mixer
Siliconized glass circle cover slides (Hampton Research)
24‐well plastic tray (Hampton Research)
Light microscope
Nylon loops for harvesting and flash‐freezing crystals (Hampton Research)
Liquid nitrogen

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Figures

Figure 7.15.1 Chemical structures of the (A ) Cy5 and (B ) Cy3 dyes. Panel A depicts a conjugate between Cy5 and the 5′‐terminal residue of an oligo‐2′‐deoxynucleotide via a phosphodiester moiety.

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Figure 7.15.2 (A ) Micrograph of a crystal of the complex between sc Pol η and a DNA primer‐template duplex with a 5′‐Cy5‐labeled primer strand. (B ) A color‐labeled crystal of the complex inside a cryoloop, ready for data collection. The scale bar at the bottom left represents 50 µm.

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Figure 7.15.3 Electron density maps for the complex between sc Pol η_1‐513 and a DNA duplex containing the 5′‐Cy5‐labeled primer are consistent with partially ordered DNA. Superimposed Fourier (2F o ‐F c ) sum (blue) and (F o ‐F c ) difference electron density (red) maps drawn at the 1σ level reveal only a tetranucleotide fragment (left) that maps to the central part of the DNA template strand. Virtually no density existed in the region presumably occupied by the primer strand or at the active site (not shown). Conversely, the protein portion is well ordered and fully surrounded by sum electron density. Carbon, nitrogen, and oxygen atoms of sc Pol η are colored in green, blue, and red, respectively. Diffraction data of high completeness with a resolution of 1.92 Å were collected on the LS‐CAT 21‐ID‐F beamline, Advanced Photon Source, Argonne National Laboratory (Argonne, Illinois). The maps were computed at a stage of the refinement with values for R‐work and R‐free of 27.1% and 30.2%, respectively.

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Figure 7.15.4 Cryoloop with a crystal of the complex between sc Pol η and a DNA primer‐template duplex with a 5′‐TAMN‐labeled primer strand. The purple tint of the crystal indicates presence of both protein and DNA. The scale bar at the bottom left represents 50 µm.

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Literature Cited

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