Abstract
The CspE protein from Escherichia coli K12 is a single-stranded nucleic acid-binding protein that plays a role in chromosome condensation in vivo. We report here that CspE binds to single-stranded DNA containing 6 or more contiguous dT residues with high affinity (K(D) < 30 nM). The interactions are predominantly through base-specific contacts. When an oligonucleotide contains fewer than 6 contiguous dT residues, the CspE interactions with single-stranded DNA are primarily electrostatic. The minimal length of single-stranded DNA to which CspE binds in a salt-resistant manner is eight nucleotides. We also show that CspE exists as a dimer in solution. We present a possible mechanism to explain the role of CspE in chromosome condensation in vivo by CspE binding to distant DNA regions in the chromosome and dimerizing, thereby condensing the intervening DNA.
Highlights
The CspE protein from Escherichia coli K12 is a singlestranded nucleic acid-binding protein that plays a role in chromosome condensation in vivo
We report here that CspE binds to single-stranded DNA containing 6 or more contiguous determined for (dT) residues with high affinity (KD < 30 nM)
CspE-single-stranded DNA (ssDNA) Binding as Monitored by Gel Filtration—We wanted to reinvestigate ssDNA binding by CspE since many previous reports were carried out at high protein concentrations (200 M [17]), high ratios of CspE to nucleic acid (between 20 and 600 CspE monomers to one nucleic acid molecule [17, 20, 28]), or both [17]
Summary
Nucleic Acids and Purification of CspE—Synthetic oligonucleotides were purchased as high pressure liquid chromatography-purified molecules from Integrated DNA Technologies. CspE was purified from the E. coli strain BL21 (DE3 pLysS pCH1). PCH1 contains cspE, from the AUG start codon to 11 bp downstream of the TAA stop codon, in the NdeI and BamHI sites of pET9a (Promega). Cells were grown at 37 °C in LB with 30 g/ml kanamycin and 34 g/ml chloramphenicol to an A600 ϭ 0.8. Cells were induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 2 h, centrifuged at 4000 ϫ g for 20 min at.
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