Biological Activity and a Partial Amino‐Acid Sequence of Escherichia coli DNA‐Binding Protein I Isolated from Overproducing Cells

Abstract

DNA-binding protein I from Escherichiu coli was purified from cells carrying the ssb gene on a multicopy plasmid. In comparison to the strain without the recombinant plasmid the DNA-binding protein was overproduced more than 20-fold. The amount of the protein was measured after the purification steps by gel electrophoresis and radial immunodiffusion. The protein was purified to homogeneity and was active in replication assays like the wild-type DNA-binding protein. The assays were enzymatic replication of single-stranded and double-stranded fd DNA. E. coli DNA-binding protein I was further subjected to amino acid sequence analysis. A monomer of the protein consists of 187 residues which correspond to a molecular weight of 19715 with 5 "/, error in analysis. The sequence of the amino-terminal 40 residues was determined and includes several basic residues of the protein. Sequence comparison between the DNA-binding protein I from E. coli and that coded by bacteriophage fd reveals similarities suggesting that DNA-binding protein I may use amino-terminal residues for binding to DNA like the phage protein.The important role of Escherichia coli DNA-binding protein I in DNA replication has been shown in vivo and in vitro and its features have been reviewed elsewhere [l]. A mutation in the gene for the protein (ssb-I) renders the cells temperature-sensitive for chromosomal DNA replication and for the growth of the 4X174-like phage ST1 [2]. The binding protein enhances chain elongation by E. coli DNA polymerase I11 holoenzyme [3] and supports strand separation by E. coli rep protein [4,4a]. It guides the initiation of conversion of phage fd single strand to replicative form (RF) to a specific site, and terminates the transcription by E. coli RNA polymerase at the origin of phage fd replication [5]. These properties suggest that E. coli DNA-binding protein I has an important role for chain growth in the replication fork of the bacterial chromosome.Cells carrying the ssb gene on a plasmid [6] produce more E. coli DNA-binding protein I than wild-type cells [7]. In this paper we confirmed the increased DNA-binding protein synthesis in the former cells by measuring its concentration in all steps of purification. The amino acid composition and a partial amino acid sequence of the homogeneous protein was determined and coinpared with the sequence of DNA-binding sites of other binding proteins. The purified protein was assayed in enzymatic phage fd viral strand and complementary strand DNA replication. MATERIALS AND METHODS StrainsEscherichia coli strain CSR603 uvrA6, recAI has been described [6]. This strain carrying the plasmid pDR2000, i.e.Abbreviations. RF, replicative form ; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulphonic acid; ssDNA and dsDNA, single-stranded and double-stranded DNA.pBR322 with a 9000-residue insertion which includes the genes uvrA and ssb-1, was kindly provided by Drs A. Sancar and W. D. Rupp [6]. BuffersBuffer A is 20 % glycerol, 50 mM Tris/HCl, pH 6.9, 1 mM dithiothreitol, 1 niM EDTA, 100 mM NaC1. B...

The single-stranded DNA-binding protein of Escherichia coli.

“…Beyreuther et al (38) sequenced the first 40 amino acids from the NH2 terminus of SSB by using automated Edman degradation. The complete amino acid sequence, however, was deduced from the DNA sequence first reported by Sancar et al (333).…”