“The Single-stranded binding proteins (SSB) hold the ssDNA and facilitate DNA replication by providing ssDNA to DNA polymerase.”


 The process of replication is not a complex one as like transcription and translation. Replication is an enzyme-dependent catalytic reaction which replicates DNA and sends it to the newly synthesized daughter cells. 

It starts with the synthesis of single-stranded RNA primers with the help of primase. Once the process of primer settling done, the helicase starts unwinding the dsDNA. However, the ssDNA is thermodynamically unstable and it is more prone to nucleophilic attack that can damage the DNA. 

This problem is solved by the nucleoprotein called SSB, single-stranded binding proteins. In the present article, we are going to discuss on the SSB,  a single-stranded binding protein and its function is replication as well as in molecular genetics. 

Before that read the previous article of this series:

  1. Meet DNA Primase: The Initiator Of DNA Replication
  2. What Is Helicase? And How It unwinds DNA? 
  3. What Is DNA Ligase? And How T4 DNA Ligase Works?

We are here, 

Single stranded binding proteins

What is a single-stranded binding protein(SSB)? 

The SSB protein is a class of DNA binding proteins that holds the single-stranded DNA to facilitate DNA replicationIn bacteria, especially in the E.Coli, the SSB protein found as a tetramer with four different domains or structures. 

All the subunits are identical and having a molecular weight of 19KDa. 

However, the commercially available single-stranded binding proteins are of approximately ~18 to 18.9KDa in size. 

Structure of SSB:

There is at least one DNA binding oligonucleotide/oligosaccharide binding (OB) fold present in it. The OB-fold contains at least five stranded beta-sheet which is arranged as a beta-barrel and then capped by a single alpha helix.

The arrangement of OB-fold is different between bacteria, archaea, and eukaryotes. 

The tetrameric structure of single stranded binding protein and its location on ssDNA.

The tetrameric structure of a single-stranded binding protein and its location on ssDNA.

Types of SSB protein:

Viral SSB protein:

ICP8, which is the SSB protein present during DNA replication of herpes simplex virus type-1 (HSV-1), is similar to human cytomegalovirus (HHV). It is one of the seven proteins encoded in the viral genome of HSV-1 for DNA replication.

It recombines to ssDNA and even melts dsDNA. During initial replication, it destabilizes dsDNA. Here, helicase and viral SSB protein are different because of the absence of ATP and Mg2+.

When any cells are infected by HSV-1, then the DNA becomes colocalized with ICP8.

The structure of ICP8 consists of a 5-stranded beta-sheet and two alpha-helices in front of the neck, 8 alpha-helices in the head, and a 3-stranded beta-sheet in the backside. ICP8 gene of HSV-1 being a nucleoprotein is present for viral DNA replication during lytic infection.

Bacterial SSB protein:

The domains of SSB protein present in bacteria are essential for DNA metabolism. It consists of three beta-strands with a single six-stranded beta-sheet to form a dimer.

Read more: Prokaryotic DNA replication.

Mitochondrial SSB protein:

Mitochondria of yeast and eukaryotes encode its own SSB protein. In yeast, mtSSB protein is encoded by the RIM1 gene while human mtSSB protein is encoded by the SSBP1 gene.

In Saccharomyces cerevisiae, mtSSB protein coordinates replication and maintenance of mtDNA. It is believed that RIM1 is exclusively responsible for ssDNA binding and can form homo-tetramers in solution.

Human mtSSB protein binds to single-stranded mtDNA as a tetramer. It has been noted that the sequence of human mitochondrial SSB is similar to Escherichia coli (EcSSB) SSB.

Interestingly, It is found that Mtp1/mtTBP, a Rim1 homolog in Candida parapsilosis which functions as a general SSB and as a sequence-specific SSB for the telomeric ends of its mitochondrial DNA, forms a significant fraction of dimmers in contrast to the stable formation of tetramers in solution.

Eukaryotic SSB:

Often called as replication protein A.

In recent studies, it has been found that replication protein present in the nucleus of eukaryotic cells is functionally similar to SSB protein but sequence homology is absent. As the name suggests, single-strand DNA binding (SSB) protein binds to single-stranded DNA. 

SSBs are a type of nucleoproteins that are present in viruses, bacteria, and humans. 

It is of utmost interest to know that the SSBs present in three branches of organisms and in viruses have the same sequences; biochemical and structural characteristics. 

They have a high affinity towards single-stranded DNA as compared to double-stranded DNA. 

Now the question arises in mind that why SSB is required? Because the helicase already has done a work to unwind the DNA. 

The answer is here,

The dsDNA structure is more stable than the ssDNA form. Three hydrogen bonds between guanine and cytosine and two hydrogen bond between the thymine and adenine make the duplex DNA more stable. 

Read more on DNA: DNA story: The structure and function of DNA

The SSB holds the ssDNA and prevents the rewinding of two ssDNA. 

In addition to this,

When SSB protein bind to single-stranded DNA, the hardening of strands is prevented during DNA replication. It also protects from the nuclease or chemical digradation.  Interestingly, it removes unwanted secondary structures for easy access to other enzymes as well. 

The SSB also helps in the binding of other proteins that are involved in DNA metabolism. Thus, SSB protein stabilizes the single-stranded DNA structures which are important for genomic progression.

The important function of SSB protein is to destabilize helical duplexes so that DNA polymerase can easily hold on to the DNA during DNA metabolism i.e. DNA replication, recombination, and repair. This is not the only function of SSB protein.

In recent years, a variety of applications of SSB protein has been found in molecular biology and analytical methods which are detailed here.

Applications of SSB in molecular genetics: 

The single-stranded binding protein is not only an in vivo separator but also it is used in some of the molecular genetic techniques such as PCR and DNA sequencing to enhance the reaction efficiency. 

With extensive research, numerous applications of SSB protein and analytical methods are now known in molecular biology.

SSB is used in DNA replication and recombination in vivo. As discussed earlier, the SSB protein holds ssDNA by destabilizing helical duplexes. This property is used to visualize ssDNA by electron microscopy. 

When SSB protein is used in PCR, the denatured DNA is stabilized and also protects the DNA from being digested by nuclease. Thus the genome can be protected. 

It has been studied that SSB protein, gene 32 protein from bacteriophage T4 or SSB from Escherichia coli is used in PCR to increase amplification efficiency with diverse templates.

SSB may also stimulate specific DNA polymerases used in DNA sequencing reactions and has been used to target restriction endonuclease digestions to specific sites in single-stranded DNA for subsequent mutagenesis.

SSB has been shown to be effective in fluorescence polarization assays and eliminates pausing when sequencing through the strong secondary structure. More recently, SSB was used to help obtain longer read lengths in pyrosequencing for SNP analysis.

When SSB is combined with RecA protein, sequences from libraries of double-stranded DNA can be used to carry out site-directed mutagenesis.

Using surface plasma resonance imaging, the affinity of SSB towards ssDNA is utilized for the detection of hybridization on the gold surface.

Apart from these, the SSB protein also functions as

  1. Protecting the genome
  2. Plays a role in maintaining telomere length

Conclusion:

The single-stranded binding protein is very necessary for the DNA polymerase to do the synthesis, if it is not there, the progression of DNA synthesis can be interrupted. Furthermore, 5 µg/µL SSB is required to enhance the amplification efficiency in PCR.