title: Iterative optimization of DNA duplexes for crystallization of SeqA-DNA complexes.
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 replication. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures. 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 DNA. 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 length and DNA ends (blunt or overhang). 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.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University.
authors.forename: Yu Seon
authors.initials: YS
authors.lastname: Chung
authors.forename: Alba
authors.initials: A
authors.lastname: Guarné
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003460:Crystallization; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009690:Nucleic Acid Conformation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; D004247:DNA