Single stranded binding protein

The single stranded DNA binding protein (SSB) of E. coli plays an important role in three aspects of DNA metabolism – namely in replication, repair and recombination. During DNA replication, SSB molecules bind to the newly separated DNA strands, keeping the strands separated by holding them in place so that each strand can serve as a template ^[1].

SSB proteins have been identified in organisms from viruses to humans. The only organisms known to lack them are Thermoproteales, a group of extremophile archaea, where they have been displaced by the protein ThermoDBP. While many phage and viral SSBs function as monomers and eukaryotes encode heterotrimeric RPA (Replication Protein A), the best characterized SSB is that from the bacteria E. coli which, like most bacterial SSBs exists as a tetramer. Active E. coli SSB is composed of four identical 19 kDa subunits. Binding of single-stranded DNA to the tetramer can occur in different "modes", with SSB occupying different numbers of DNA bases depending on a number of factors, including salt concentration. For example, the (SSB)₆₅ binding mode, in which approximately 65 nucleotides of DNA wrap around the SSB tetramer and contact all four of its subunits, is favoured at high salt concentrations in vitro. At lower salt concentrations, the (SSB)₃₅ binding mode, in which about 35 nucleotides bind to only two of the SSB subunits, tends to form. Further work is required to elucidate the functions of the various binding modes in vivo.

SSB consists is a homotetramer that has a DNA binding domain which binds to a single strand of DNA. The tetramers consist of α-helices, β-sheets, and random coils. Each subunit contains an and several . The secondary structure also includes a NH2 terminus, which consists of multiple positively charged amino acids. The DNA-binding domain lies within 115 amino acid residues from this terminus ^[2]. The COOH terminus includes many acidic amino acids.

Binding Interactions in the Active Site

ssDNA can interact with binding proteins through hydrogen bonds, stacking, or electronegative interactions. Most interactions between SSB and ssDNA happen through the OB fold. OB stands for oligosaccharide/oligonucleotide binding site. This fold consists of a 5-stranded β barrel that ends in an α-helix. is an important DNA binding site. It has been shown to be the site for cross-linking. Tryptophan and Lysine residues are important in binding as well. Treatments resulting in modification of arginine, cysteine, or tyrosine residues had no effect on binding of SSB to DNA, whereas modification of either lysine residues (with acetic anhydride) or tryptophan residues (with N-bromosuccinimide) led to complete loss of binding activity ^[3]. The two tryptophan residues involved in DNA binding are Trp40 and Trp54, which was determined by mutagenesis. One more binding site was determined by site-specific mutagenesis. When His55 is substituted with Leu it decreases binding affinity. All of these residues are found in a hydrophobic region, which is suitable for nucleotide base interactions.

SSB-Protein Interactions

SSB can form complexes with many other proteins. This trait can keep enzymes needed for damage repair, transcription, etc. near the ssDNA and it is thought that SSB can even help to stimulate these enzymes to carry out their jobs. When DNA binds SSB, most of the molecule loses flexibility. But the COOH terminal domain remain flexible, even after DNA binding. It is believed that the COOH terminus has something to do with protein binding ^[4].

SSB has also been thought to bind with exonuclease I, DNA polymerase II,

and a protein n, which is used to help synthesize RNA primers for the lagging strand. SSB can also help regulate transcription by competing with other proteins for binding spaces on DNA. SSB has a higher affinity for DNA than most other proteins, and those proteins are not able to remove SSB from DNA and bind themselves. This type of mechanism can not only regulate transcription, but it can provide protection for the DNA ^[5].

Phe60 is an important DNA binding site. It has been shown to be the site for cross-linking.

and residues are important in binding as well. Treatments resulting in modification of arginine, cysteine, or tyrosine residues had no effect on binding of SSB to DNA, whereas modification of either lysine residues (with acetic anhydride) or tryptophan residues (with N-bromosuccinimide) led to complete loss of binding activity ^[6]. The two tryptophan residues involved in DNA binding are and Trp54, which was determined by mutagenesis. One more binding site was determined by site-specific mutagenesis. When His55 is substituted with Leu it decreases binding affinity. All of these residues are found in a hydrophobic region, which is suitable for nucleotide base interactions.

SSB-Protein Interactions

Most of the molecule loses flexibility after DNA binding. But three of the phenylalanines (147, 171, 177) in the COOH terminal domain remain flexible, even after DNA binding. It is believed that the COOH terminus has something to do with protein binding ^[7]. One experiment in which Phe-177 was changed to Cys resulted in a protein that could not replicate DNA. This replication defect results from the inability of C-terminus to bind to other replication proteins ^[8].

It is believed that may play an important role in binding the RecA protein. Mutations in Gly15 have

extreme effects on recombinational repair. SSB has also been thought to bind with exonuclease I, DNA polymerase II, and a protein n, which is used to help synthesize RNA primers for the lagging strand.