The authors wish to thank the PXRR staff at the NSLS (Brookhaven National Laboratory) for assistance during data collection and Monica Pillon for help with DNA purification. This work was supported by the Canadian Institutes of Health Research (MOP 67189).

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 (SeqA&Delta;(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 ...

<ol>
	<li>Campbell, J. L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+oriC+and+the+dnaA+gene+promoter+are+sequestered+from+dam+methyltransferase+following+the+passage+of+the+chromosomal+replication+fork.">E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.</a> Cell. 62, 967-979 (1990).</li><li>Guarn&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+SeqA-N+filament:+implications+for+DNA+replication+and+chromosome+organization.">Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.</a> Embo. J. 24, 1502-1511 (2005).</li><li>Brendler, T., Austin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+SeqA+protein+to+DNA+requires+interaction+between+two+or+more+complexes+bound+to+separate+hemimethylated+GATC+sequences.">Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.</a> Embo. J. 18, 2304-2310 (1999).</li><li>Jordan, S. R., Whitcombe, T. V., Berg, J. M., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+variation+in+DNA+length+yields+highly+ordered+repressor-operator+cocrystals.">Systematic variation in DNA length yields highly ordered repressor-operator cocrystals.</a> Science. 230, 1383-1385 (1985).</li><li>Tan, S., Hunziker, Y., Pellegrini, L., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+the+yeast+MATalpha2/MCM1/DNA+ternary+complex:+general+methods+and+principles+for+protein/DNA+cocrystallization.">Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.</a> J. Mol. Biol. 297, 947-959 (2000).</li><li>Rice, P. A., Yang, S., Mizuuchi, K., Nash, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+IHF-DNA+complex:+a+protein-induced+DNA+U-turn.">Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn.</a> Cell. 87, 1295-1306 (1996).</li><li>Yang, W., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+site-specific+recombinase+gamma+delta+resolvase+complexed+with+a+34+bp+cleavage+site.">Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site.</a> Cell. 82, 193-207 (1995).</li><li>Chung, Y. S., Brendler, T., Austin, S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+cooperative+binding+of+SeqA+to+a+tandem+GATC+repeat.">Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.</a> Nucleic Acids Res. , (2009).</li><li>Anderson, J., Ptashne, M., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocrystals+of+the+DNA-binding+domain+of+phage+434+repressor+and+a+synthetic+phage+434+operator.">Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 1307-1311 (1984).</li><li>Timsit, Y., Moras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+self-fitting:+the+double+helix+directs+the+geometry+of+its+supramolecular+assembly.">DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.</a> Embo J. 13, 2737-2746 (1994).</li><li>Chung, Y. S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+SeqA+bound+to+a+pair+of+hemimethylated+GATC+sites.">Crystallization and preliminary X-ray diffraction analysis of SeqA bound to a pair of hemimethylated GATC sites.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 64, 567-571 (2008).</li><li>DeLano, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphic Systems. , (2002).</li></ol>

No conflicts of interest declared.

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...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue #</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>TRIS</td>
 <td>Bioshop</td>
 <td>TRS003.5</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher Scientific</td>
 <td>E478-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiotheitrol (DTT)</td>
 <td>Bio Basic Inc.</td>
 <td>DB0058</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Bioshop</td>
 <td>SOD002.10</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Caledon</td>
 <td>5350-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>S5016-500G</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Bioshop</td>
 <td>SDS001.500</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Bioshop</td>
 <td>URE001.5</td>
 <td></td>
 </tr>
 <tr>
 <td>40% 29:1 Bis/acrylamide</td>
 <td>Bio Basic Inc.</td>
 <td>A0007-500ml</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Boric acid</td>
 <td>EMD</td>
 <td>BX0865-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylene cyanol FF</td>
 <td>Bio-Rad</td>
 <td>161-0423</td>
 <td></td>
 </tr>
 <tr>
 <td>Bromophenol Blue</td>
 <td>Bioshop</td>
 <td>BR0222</td>
 <td></td>
 </tr>
 <tr>
 <td>Dual Adjustable Vertical Gel System</td>
 <td>C.B.C. Scientific Company Inc.</td>
 <td>DASG-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Index crystallization screen</td>
 <td>Hampton Research</td>
 <td>HR2-144</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Wizard I crystallization screen</td>
 <td>Emerald BioSystems</td>
 <td>EBS-WIZ-1</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Wizard II crystallization screen</td>
 <td>Emerald BioSystems</td>
 <td>EBS-WIZ-2</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Classics crystallization screen</td>
 <td>Qiagen</td>
 <td>130701</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Intelliplate trays </td>
 <td>Art Robbins Instruments</td>
 <td>102-0001-00</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>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 &deg;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 &deg;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 &deg;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.</td>
 </tr>
 </tbody>
 </table>

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

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 (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

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

Crystal structure of protein&#x2013;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.

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

iterative-optimization-dna-duplexes-for-crystallization-seqa-dna

Research

JoVE Journal

Biology

9.9K visualizações.  McMaster University. Estrutura cristalina de complexos DNA-proteína pode fornecer informações sobre a função da proteína, mecanismo, assim como, a natureza da interacção específica. Aqui, nós relatamos como otimizar o comprimento, a sequência e as extremidades do DNA duplex para co-cristalização com Escherichia coli SEQA, um regulador negativo da iniciação da replicação.