Interaction of E. coli Single-Stranded DNA Binding Protein (SSB) with Exonuclease I. The Carboxy-Terminus of SSB Is the Recognition Site for the Nuclease
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Jochen Genschel
Abstract
The 3[']-5['] singlestranded DNA (ssDNA) degrading exonuclease I of E. coli directly interacts with the E. coli ssDNA binding protein (EcoSSB). Analytical ultracentrifugation shows that all 4 carboxytermini of an EcoSSB tetramer bind exonuclease I. Binding is weakened by increasing salt concentrations, indicating the involvement of the negatively charged amino acids of the carboxyterminus of SSB. Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSSBF177C do not bind to exonuclease I while EcoSSBG15D (ssb-3) does bind. In a coprecipitation assay we show that the absence of the last ten amino acids (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I. The interaction does not depend on the presence of the correct aminoterminal DNA binding domain or the amino acid sequences between the DNA binding domain and the last ten amino acids. A synthetic peptide (WMDFDDDIPF), corresponding to the last nine amino acids of EcoSSB, specifically inhibits the interaction. Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wildtype EcoSSB, indicating that the phenotype of ssb-113 is not an indication of altered DNA binding. The repair deficiency of either ssb-3 or ssb-113 strain can be complemented by overexpression of the respective other mutant.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- Interaction of E. coli Single-Stranded DNA Binding Protein (SSB) with Exonuclease I. The Carboxy-Terminus of SSB Is the Recognition Site for the Nuclease
- p38/SAPK2-Dependent Gene Expression in Jurkat T Cells
- The First Laminin G-Type Domain in the SHBG-Like Region of Protein S Contains Residues Essential for Activation of the Receptor Tyrosine Kinase Sky
- Permutation of the Active Site Motif of Tryparedoxin 2
- Structural Investigations of the Highly Flexible Recombinant Ribosomal Protein L12 from Thermotoga maritima
- Polyhistidine-Tagged Hepatitis B Core Particles as Carriers of HIV-1/gp120 Epitopes of Different HIV-1 Subtypes
- In Vitro Evolution of Ligands for HCV-Specific Serum Antibodies
- Stability of Bacteriophage T4 Short Tail Fiber
- Analysis of the RNase T1 Mediated Cleavage of an Immobilized Gapped Heteroduplex via Fluorescence Correlation Spectroscopy
- Binding of Selenoprotein P to Heparin: Characterization with Surface Plasmon Resonance
- Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases
