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== Sandbox Single Stranded DNA-Binding Protein (SSB)==
<StructureSection load='1eyg' size='400' side='right' frame='true' caption='E. coli single-stranded DNA-binding protein chymotryptic fragment complex with DNA (PDB code [[1eyg]])' scene=''>
<StructureSection load='1eyg' size='500' side='right' frame='true' align='right' caption='Structure of Single Stranded DNA-Binding Protein bound to ssDNA (PDB entry [[1eyg]])' scene=''>
Single Stranded DNA-Binding Protein (SSB)
==Overview==
'''Single-stranded DNA-binding protein''' '''(SSB)''' binds to single-stranded regions of DNA.  This binding serves a variety of functions - it prevents the strands from hardening too early during replication, it protects the single-stranded DNA from being broken down by nucleases during repair, and it removes the secondary structure of the strands so that other enzymes are able to access them and act effectively upon the strands<ref>PMID:2087220</ref>. 


==Introduction==
Single-stranded DNA (ssDNA) is utilized primarily during the course of major aspects of DNA metabolism such as replication, recombination and repair <ref>PMID: 2087220</ref>. In addition to stabilizing ssDNA, SSB proteins also bind to and control the function of many other proteins that are involved in all three of these major DNA metabolic processes. During DNA replication, SSB molecules bind to the newly separated individual DNA strands, keeping the strands separated by holding them in place so that each strand can serve as a template for new DNA synthesis<ref>Berg JM, Tymoczko JL, Stryer L. ''Biochemistry''. 6th edition. New York: W H Freeman; 2006.</ref>.
The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.


Copies of SSB bind to the unwound DNA strands, keeping the strands separated so that both strands can serve as templates.
==Structure of ''E. coli'' SSB==


==Structure==


The structure of SSB consists of a homotetramer that has a DNA binding domain that binds to a single strand of DNA.  
SSB proteins have been identified in many different organisms, but the most well understood SSB remains the SSB of ''E. coli''.  ''E. coli'' SSB is a homotetramer consisting of <scene name='56/566528/Homotetramer/1'>four identical subunits</scene> which are each about 19 kDa in size <ref>PMID:2087220</ref>.  There are two different binding modes of the ''E. coli'' SSB when it complexes with ssDNA<ref>PMID:11993998</ref>.  Regulation of these modes has been found to be dependent on salt concentration, in addition to other unknown factors.  Under low salt conditions, the protein is less efficient as only two of the four identical subunits of ''E. coli'' SSB were found to bind to the ssDNA <ref>PMID:11993998</ref>. This is a common theme among DNA binding proteins. The cause is presumed to be that the protein has less ion decoration at lower salt levels. And it could be that the subunits interact with each other through salt bridges to remain close to each other and the DNA. Under high salt concentrations, however, all four subunits of the homotetramer bind to the ssDNA, increasing the number of nucleotides in contact with the SSB and thus favoring SSB-ssDNA interactions.  Depending on the salt concentration and other factors, estimates of the size of the site of interaction between SSB and ssDNA range anywhere from 30 to 73 nucleotides for each tetramer <ref>PMID:11993998</ref>. 
 
Active ''E. coli'' SSB is made of a homotetramer with extensive DNA binding domains that bind to <scene name='56/566528/Dna/1'>a single strand of DNA</scene><ref>PMID:2087220</ref>. The tetramers consist of <scene name='56/566528/E_coli_ssb_alpha_helices/1'>α-helices</scene>, <scene name='56/566528/Beta_sheets/2'>β-sheets</scene>, and random coils. Each subunit contains an α-helix and several β-sheets. The secondary structure also includes a NH2 terminus, which consists of multiple basic residues, or <scene name='56/566528/Basic_residues/2'>positively charged amino acids</scene>.  The DNA-binding domain lies within 115 amino acid residues from this terminus.  The COOH terminus includes many negatively charged, or <scene name='56/566528/Acidic_residues/5'>acidic amino acids</scene> <ref>PMID:2087220</ref>.


==Binding Interactions in the Active Site==
==Binding Interactions in the Active Site==


Residues involved in ssDNA binding
<scene name='56/566528/Ssdna/1'>Single-stranded DNA</scene> can interact with SSB through hydrogen bonds, stacking, or electrostatic interactions.  Though SSB proteins are found in a variety of different organisms, most interactions between SSB and ssDNA happen through the common structural motif of an oligosaccharide/oligonucleotide binding site, referred to as the <scene name='56/566528/Ob_fold/4'>OB fold</scene> <ref>Shamoo, Yousif.  “Single Stranded DNA binding proteins.” ‘’Encyclopedia of Life Sciences.’’  MacMillan Publishers Ltd, Nature Publishing Group; 2002</ref>. The OB fold allows SSB to bind preferentially to ssDNA. Each subunit of a SSB has an <scene name='56/566528/Ob_fold/1'>OB fold</scene> (the SSB of E. coli thus has <scene name='56/566528/Ob_fold/2'>four OB folds</scene>, one per each of its <scene name='56/566528/Homotetramer/1'>four identical subunits</scene>). This fold consists of a <scene name='56/566528/Beta_barrel/1'>5 stranded β barrel</scene> that ends in an <scene name='56/566528/Beta_barrel/2'>α-helix</scene>. 
The Trp fluorescence of SSB is quenched by approx90% upon binding poly(dT) in the (SSB)65 binding mode5, 6. However, in the (SSB)35 mode, Trp fluorescence is quenched by only approx50% due to the fact that only half of the Trp residues interact with DNA in this binding mode11 (Fig. 4b). Spectroscopic studies also suggest that Trp 40 and Trp 54 form stacking interactions with the bases13, 14. Mutagenesis of Trp 40 and Trp 54 reduce ssDNA binding affinity14, 15. Crosslinking experiments16 and mutational studies17, 18 have shown that Phe 60 is involved in DNA binding. Consistent with these observations, Trp 40, Trp 54 and Phe 60, in the structure of the SSBc−ssDNA complex, make extensive interactions with the ssDNA (see above).


Only a few natural ssb mutants have been isolated and characterized; two of them, ssb-1 and ssb-3, are located within SSBc. The ssb-1 mutant, a H55Y mutation, destabilizes the SSB tetramer to favor monomers1, 2, 12. The ssb-3 mutant, a G15D mutation, renders the cell extremely sensitive to UV19. In the model of the SSBc−ssDNA structure presented here, Gly 15 is within 3.5 Å of the phosphate backbone at C4 (Fig. 2a). Mutation to Asp may sterically hinder ssDNA binding. The structure therefore suggests that G15D would affect ssDNA binding.
Several specific amino acid residues play essential roles in the binding of ssDNA to SSB. <scene name='56/566528/Phe_60/2'>Phe60</scene> is a key residue involved in binding the ssDNA to the protein, as it has been shown to be the site for cross-linking. Tryptophan and Lysine residues are important in binding as well, as evidenced by modification treatments of lysine and tryptophan residues resulting in a complete loss of binding activity for the protein. The two tryptophan residues involved in ssDNA binding are <scene name='56/566528/Trp_40_and_trp_54/1'>Trp40 and Trp54</scene>, which were determined by mutagenesis <ref>PMID:2087220</ref>.


Finally, thermodynamic studies indicate that electrostatic interactions have a major role in SSB−ssDNA binding11, 20. The role of Lys residues and the N-terminus in ssDNA binding has also been probed by chemical modification21 and it was observed that acetylation of Lys 43, Lys 62, Lys 73, Lys 87, and the terminal amine is greatly reduced upon binding ssDNA. In the structure, these Lys residues, as well as the N-terminal amine, are within contact distance of the ssDNA backbone and selective acetylation of these residues would be expected to have a significant effect on ssDNA binding. Other basic residues make interactions with the ssDNA, either with the ssDNA bases (Arg 3) or with the phosphate backbone (Arg 84).
One more key residue in the binding site, <scene name='56/566528/His_55/2'>His55</scene>, was determined by site-specific mutagenesis, as when <scene name='56/566528/His_55/2'>His55</scene> is substituted with Leu it decreases the overall binding affinity for ssDNA. All of these residues are found in a <scene name='56/566528/Hydrophobic_region/2'>hydrophobic region</scene>, which is suitable for nucleotide base interactions. Treatments that modified arginine, cysteine, or tyrosine residues had no effect on binding of SSB to DNA, suggesting that these amino acids are not involved in significant interactions of the protein with the ssDNA.  


Overall, the chemical composition of the protein−ssDNA interface is mixed and a similar number of interactions are made between the protein and the bases or the protein and the ssDNA backbone.
==Interactions Between ''E. coli'' SSB and other Proteins==


The (SSB)65 binding mode
Most of the molecule loses flexibility after ssDNA binding. However, three phenylalanine residues in the COOH terminal domain remain flexible, even after DNA binding, suggesting that the COOH terminus has something to do with protein binding <ref>PMID:2087220</ref>. An experiment where one of the three Phe residues was changed to Cys resulted in a protein that could not replicate DNA. This replication defect stemming from the lost phenylalanine residue was likely a result of the inability of the altered C-terminal region to bind other proteins necessary for replication <ref>PMID:2453719</ref>.
From the structure of SSBc bound to the two molecules of dC(pC)34, a model can be proposed for how a continuous stretch of ssDNA can interact with all four subunits and wrap around the tetramer. Such a model can be generated simply by applying the D2 symmetry operators (which relate the four subunits together) to the dC[pC]3−30 fragment to fill the gaps existing between the dC[pC]3−30, dC[pC]3−16, and dC[pC]19−27 ssDNA fragments. A model for SSBc bound to a long continuous ssDNA is presented in Fig. 4a. The proposed structural model for the (SSB)65 binding mode recapitulates remarkably well most of the biochemical and biophysical properties of this binding mode. First, the length of ssDNA occluded by the SSB tetramer is 65 nucleotides2. Second, ssDNA interacts with all four subunits, consistent with equilibrium fluorescence binding measurements2. Third, the ssDNA wraps around the outside of the tetramer7.


The (SSB)35 binding mode
<scene name='56/566528/Gly_15/2'>Gly15</scene> is believed to play an important role in binding the RecA protein. SSB will interact with the protein RecA to enable recombination, because RecA will recognize SSB and replace it on the strand. In DNA repair, SSB will bind to the damaged strand to protect it. And eventually it will attract repair enzymes which will replace SSB and begin repair mechanisms. Mutations in <scene name='56/566528/Gly_15/2'>Gly15</scene> have been shown to have extreme effects on recombinational repair. SSB is also thought to bind with exonuclease I, DNA polymerase II, and a protein n, which is a part of the primosome complex and used to help synthesize RNA primers for the lagging strand <ref>PMID: 2087220</ref>.
A model for the (SSB)35 binding mode can also be postulated based on the structure of the SSBc−ssDNA complex by using the symmetry related complexes generated along the L45 loops. Note that the role of the L45 loop residues in intertetramer cooperativity has not been investigated in solution, and that evidence for the use of a tetramer−tetramer interface involving the L45 loops is uniquely crystallographic12 (also see above). Once the symmetry related SSBc−ssDNA complex structures that occur within the crystal are displayed, it becomes apparent that there is one path by which a long continuous stretch of ssDNA can interact with two adjacent SSBc tetramers to form a ssDNA bound filament of SSBc. This path is described in Fig. 4b,c. In this model, the ssDNA occupies the entire binding site of one SSBc subunit (indicated in cyan in Fig. 4b), half of the binding sites of two subunits (indicated in green and gold in Fig. 4b), and does not occupy the ssDNA binding site of the fourth subunit (in red in Fig. 4b). Although the (SSB)35 structural model is speculative, it is consistent with the biochemical information obtained for this binding mode. First, the occluded site size determined from the structural model is approx35 nucleotides per tetramer. Data from fluorescence equilibrium binding studies of SSB to oligodeoxynucleotides suggest that only two subunits of the SSB tetramer interact with ssDNA in the (SSB)35 mode2, 11. In fact, although the structural model in Fig. 4b indicates partial interactions with three of the subunits, the sum of these interactions is equivalent to those for only two subunits.


The structure of the SSBc−ssDNA complex reveals how the homotetrameric protein utilizes its perfectly symmetrical D2 structure to bind and compact a long stretch of ssDNA by wrapping it extensively around a relatively small protein. The structure also suggests a more speculative model by which the ssDNA can be compacted partially and still allow cooperative interactions between adjacent tetramers.


==See Also==
==Other SSB Structures==
<StructureSection load='2vw9' size='400' side='left' frame='true' caption='Structure of Single Stranded DNA-Binding Protein from Helicobacter pylori bound to ssDNA (PDB entry [[2vw9]])' scene=''>


Though the SSB of ''E. coli'' is perhaps the best characterized, ssDNA binding proteins of many other organisms have also been identified.  Some proteins, such as the SSB of ''E. coli'' and human mitochondrial SSBs, bind as tetramers to ssDNA.  However, the SSB can have from one to as many as four OB-fold containing subunits in its structure<ref>Shamoo, Yousif.  “Single Stranded DNA binding proteins.” ‘’Encyclopedia of Life Sciences.’’  MacMillan Publishers Ltd, Nature Publishing Group; 2002</ref>.  For example, the structure of the SSB from ''Helicobacter pylori'' shown to the left is a homodimer composed of <scene name='56/566528/Homodimers/1'>two identical subunits</scene>, each with an <scene name='56/566528/Ob_fold_helico/1'>OB-fold motif</scene> made of similar secondary structure elements such as an <scene name='56/566528/Alpha_helices/3'>α-helix</scene> and several <scene name='56/566528/Beta_sheets/1'>β-sheets</scene><ref>PMID:19285993</ref>.  Just like in the ''E. coli'' SSB, the <scene name='56/566528/Ob_fold_helico/1'>OB-fold area</scene> in each subunit is used for single-stranded nucleic acid binding<ref>Shamoo, Yousif.  “Single Stranded DNA binding proteins.” ‘’Encyclopedia of Life Sciences.’’  MacMillan Publishers Ltd, Nature Publishing Group; 2002</ref>.  A phenylalanine residue (<scene name='56/566528/Labeled_phe/1'>Phe56</scene>) is again integral to ssDNA binding, as it is a site of cross-linking.  Tryptophan and lysine residues again play an important role in binding of ssDNA to the protein. 


==References==
As single-stranded DNA binding proteins are utilized in some of the most important aspects of DNA metabolism, they are used extensively in DNA replication, repair and recombination<ref>PMID: 2087220</ref>.  Most SSBs use one or more subunits with an OB-fold motif to bind securely and preferentially to ssDNA.  A few specific SSBs (such as RecA and adenovirus DBP) do not use the OB-fold, instead relying on electrostatic and stacking interactions as well as hydrogen bonding<ref>Shamoo, Yousif.  “Single Stranded DNA binding proteins.” ‘’Encyclopedia of Life Sciences.’’  MacMillan Publishers Ltd, Nature Publishing Group; 2002</ref>. 
<references/>


PMID: 2087220


==See Also==


</StructureSection>
* [[DNA Replication, Transcription and Translation]]
* [[Translation]]
* [[Transcription]]
*[[2vw9]]
==References==
<references/>

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