Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single‐stranded DNA binding protein

Abstract

The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine. The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability. While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly (dT). For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein. The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein.In the living cell, the single-stranded DNA binding protein of Escherichia coli (EcoSSB) stabilizes the single-stranded conformation of the DNA which is necessary for subsequent reactions like replication, recombination or repair. Another important functional role of EcoSSB is the protection of single-stranded DNA against nucleolytic attack. For a review of biological and physico-chemical properties of EcoSSB seeThe amino acid sequence of EcoSSB (177 amino acids) has been deduced from its DNA sequence [3]. The protein has been crystallized [4, 51, but so far, no tertiary structure from X-ray crystallography is known. Under native conditions four identical EcoSSB protomers form a tetramer with a D2 symmetry [6]. Secondary structure prediction according to the algorithm of Chou and Fasman [7] suggests the presence of two structurally different regions in the protomer [3]. Approximately 100 amino-terminal residues form a structured region containing M helices and P sheets whereas the remaining carboxy-terminal part containing 19 glycine and 10 proline residues seems to have no definable secondary structure. It could be shown from partial proteolytic digests that removing 62 amino acids from the carboxy terminus did not diminish the DNA binding ability; it only slightly reduced the tetramerization of the protein [S].A variety of mutagenesis experiments has been carried out to elucidate the role of defined amino acids in the structure and function of EcoSSB. It could be shown that the tryptophan residues at positions 40 and 54 [9, 101 and the phenylalanine residue at position 60 [ll] are involved in DNA binding. 11, 21.We have previously shown that the phenylalanine residue at position 60 is directly involved in a hydrophobic interaction with the DNA [12]. From chemical mutagenesis a temperature-sensitive mutant strain of E. coli could be isolated in which histidine at position 55 was substituted by tyrosine (ssb-1) [13]. The temperature sensitivity of the ...

Analysis of ssb mutations in vivo implicates SSB protein in two distinct pathways of SOS induction and in recombinational DNA repair

“…Although Trp-40 and Phe-60 are highly conserved in conjugative plasmid and mitochondrial SSBs and mutations at these positions affect in vitro ssDNA binding Casas-Finet et al, 1987), only the ssb F60→S mutation had even a modest effect on DNA repair. Whereas the presence of His-55 is important for the tetrameric structure of the protein, a pattern of amino acid preference at that position has not been recognizable based on aromaticity or hydrophobicity of amino acid substitutions (Curth et al, 1991). However, our current studies of ssb H55→K intragenic suppressors may assist in understanding the structurefunction relationship of tetramerization.…”

“…Excess SSB inhibits the nucleation of RecA as demonstrated by microscopic analysis of RecA filament formation (Chrysogelos and Griffith, 1982) and reduces the rate of RecA polymerization quite extensively (Thresher et al, 1988). A reduced ssDNA-binding affinity such as that demonstrated by the ssb-1 and ssb W54→S mutations (Curth et al, 1991; would decrease the competition between SSB and RecA for ssDNA. This action could subsequently affect the length of the RecAssDNA filament during its polymerization as originally proposed by Moreau (1987;1988).…”

“…The site-directed ssb mutations were generated by Ute Curth and Claus Urbanke in the plasmid pSF1 by cassette mutagenesis (Bayer et al, 1989) or duplex-gapped mismatch-primer mutagenesis (Curth et al, 1991). The gene internal ssb fragment produced by a PpuMI-BssHII double digest was used to transfer the mutant gene fragments of ssb W54→F, ssb W54→G, ssb W54→L, ssb F60→S, ssb W88→Y, and ssb W135→T from the pSF1 plasmid into pHSG575, a pSC101 derivative containing the ssb-1 gene (refer to Carlini et al, 1993).…”

“…Structural knowledge of Eco SSB has been limited by the lack of a detailed X-ray structure because the complete SSB protein is refractory to crystallization (Ollis et al, 1983). However, site-directed mutations involving amino acids implicated in DNA binding and protein multimerization have increased our knowledge of protein structure and function including the role of the His-55 residue in protein multimerization (Bayer et al, 1989) and the involvement of the Phe-60 residue in a hydrophobic interaction with ssDNA (Curth et al, 1991). Polar amino acid substitutions at Trp-54 have additionally supported the direct interaction of this residue with ssDNA which was originally suggested by spectrophotometric analysis of stacking interactions Khamis et al, 1987).…”

Single-stranded DNA-binding Protein Recruits DNA Polymerase V to Primer Termini on RecA-coated DNA

“…Proteins-Proteins used in this study were purified as described in the respective references: RecA (23); E. coli SSB mutant proteins H55Y (SSB-1), H55K, and P176S (SSB-113) (24); polV (MBP-UmuC and UmuDЈ purified separately) (7,8); E. coli SSB (25,26), with an additional purification step of a Mono P column; the ␤ subunit of polIII (27). Human simplex virus type 1 (HSV) ICP8 was a gift from I. Robert Lehman (Stanford University School of Medicine), and the ␥ complex and human RPA were gifts from Michael O'Donnell (The Rockefeller University, New York).…”