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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=''>
'''Single-stranded DNA-binding protein''', or SSB, binds to single-stranded regions of DNA in order to prevent premature annealing, to protect the single-stranded DNA from being digested by nucleases, and to remove secondary structure from the DNA to allow other enzymes to function effectively upon it. Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination and repair. As well as stabilizing this single-stranded DNA, SSB proteins bind to and modulate the function of numerous proteins involved in all of these processes.
Single Stranded DNA-Binding Protein (SSB)
==Overview==
==Overview==
<StructureSection load='1eyg' size='400' side='right' frame='true' caption='Structure of Single Stranded DNA-Binding Protein bound to ssDNA (PDB entry [[1eyg]])' scene=''>
'''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>. 


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 <ref>PMID: 2087220</ref>.
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>.


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)<sub>65</sub> 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)<sub>35</sub> 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''.
==Structure of ''E. coli'' SSB==


</StructureSection>


==Structure==
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>. 
<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=''>


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 <scene name='56/566528/Alpha_helices/3'>α-helix</scene> and several <scene name='56/566528/Beta_sheets/1'>β-sheets</scene>. 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 <ref>PMID: 2087220</ref>. The COOH terminus includes many acidic amino acids.  
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==


<scene name='56/566528/Labeled_phe/1'>Phe56</scene> is an important DNA binding site. It has been shown to be the site for cross-linking.
<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>.
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 <ref>PMID: 2087220</ref>.
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==
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>.


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


==Binding Interactions between DNA and SSB of ''E. coli''==
==Interactions Between ''E. coli'' SSB and other Proteins==


<StructureSection load='1qvc' size='400' side='right' frame='true' caption='Structure of Single Stranded DNA-Binding Protein from E. coli (PDB entry [[1qvc]])' scene=''>
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>.
Phe60 is an important DNA binding site. It has been shown to be the site for cross-linking.
<scene name='56/566528/Tryptophan_residues/1'>Tryptophan</scene> 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 <ref>PMID: 2087220</ref>.  
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==
<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>.


It is believed that <scene name='56/566528/Gly_15/1'>Gly15</scene> 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.


</StructureSection>
==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. 
 
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>. 
 


==See Also==
==See Also==
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* [[Translation]]
* [[Translation]]
* [[Transcription]]
* [[Transcription]]
*[[2vw9]]
==References==
==References==
<references/>
<references/>

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Refayat Ahsen, Rachel Craig, Alexander Berchansky, Michal Harel
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