Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Summary

Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Abstract

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication¹. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures². SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA³. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length⁴ and DNA ends (blunt or overhang)^5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqAΔ(41-59)-A25R) to obtain crystals suitable for structure determination.

Protocol

1. Protein Purification

The flexible linker connecting the N- (oligomerization) and C-terminal (DNA binding) domains of SeqA aids the recognition of hemimethylated GATC repeats separated by one to three turns on the DNA. For this study, we used a dimeric variant of SeqA (SeqAΔ(41-59)-A25R) with a point mutation in the N-terminal domain that prevents further oligomerization and a shortened linker that restricts DNA binding to tandem GATC repeats separated exclusively by one turn on the DNA (Figure 1)^2,8.

1. Transform BL21 (DE3) cells with the plasmid encoding SeqA under the control of T7 promoter,
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Discussion

One of the biggest challenges in macromolecular X-ray crystallography is obtaining diffraction quality crystals. In the case of protein or protein-DNA complexes, this challenge is exacerbated due to the additional variables that must be optimized. It is widely believed that the length of the DNA and the presence of sticky overhangs to enhance association of neighboring DNA molecules in a longer pseudo-duplex are the main parameters to optimize. However, we have shown that the nature and length of these overhangs can have.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
TRIS	Bioshop	TRS003.5
Ethylenediaminetetraacetic acid (EDTA)	Fisher Scientific	E478-500
Dithiotheitrol (DTT)	Bio Basic Inc.	DB0058
NaCl	Bioshop	SOD002.10
Glycerol	Caledon	5350-1
Sucrose	Sigma-Aldrich	S5016-500G
Sodium dodecyl sulfate (SDS)	Bioshop	SDS001.500
Urea	Bioshop	URE001.5
40% 29:1 Bis/acrylamide	Bio Basic Inc.	A0007-500ml	Store at 4 °C
Boric acid	EMD	BX0865-1
Xylene cyanol FF	Bio-Rad	161-0423
Bromophenol Blue	Bioshop	BR0222
Dual Adjustable Vertical Gel System	C.B.C. Scientific Company Inc.	DASG-250
Index crystallization screen	Hampton Research	HR2-144	Store at 4 °C
Wizard I crystallization screen	Emerald BioSystems	EBS-WIZ-1	Store at 4 °C
Wizard II crystallization screen	Emerald BioSystems	EBS-WIZ-2	Store at 4 °C
Classics crystallization screen	Qiagen	130701	Store at 4 °C
Intelliplate trays	Art Robbins Instruments	102-0001-00
Solutions Protein purification buffer: 100 mM TRIS pH 8, 2 mM EDTA, 2 mM DTT and 5% glycerol. Protein storage buffer: 20 mM TRIS pH 8, 150 mM NaCl, 5 mM DTT, 0.5 mM EDTA and 5% glycerol. Gel loading mix: Add 20 g of sucrose, 25 mg of bromophenol blue, 25 mg of xylene cyanol FF, 1 ml of 10% w/v SDS and 10 ml of 10X TBE to 70 ml of autoclaved ddH2O. Stir with mild heating until sucrose is dissolved and adjust the final volume to 100 ml with autoclaved ddH2O. Store at 4 °C. 2X loading buffer: Add 11 g of urea to 10 ml of gel loading mix. Stir on a hot plate until urea dissolves. Aliquot in 2 ml tubes and store at 4 °C. 10X PAGE mix: Mix 420.4 g of urea, 100 ml of 10X TBE (autoclaved), 250 ml of 40% 29:1 Bis/Acrylamide in ddH2O. Stir until totally dissolved and adjust volume to 1 liter. Store in dark bottles at 4 °C. 10X TBE: Dissolve 108 g of TRIS, 55 g of boric acid and 9.3 g of EDTA in 1 liter of ddH2O. Autoclave and store at room temperature. Elution buffer: Dilute 8 ml of 5 M NaCl, 2 ml of 1 M TRIS pH 7.5, 0.4 ml of 0.5 M EDTA pH 8 on 200 ml of ddH2O. Autoclave and store at room temperature.