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E. coli SSB (Eco SSB)

- Discovery
Single-stranded DNA-binding proteins of E. coli have been discovered by Nolan Sigal and his colleagues in 1972. When this protein was discovered, it was called a DNA-unwinding protein (M.W. 22,000). According to Sigal, it binds to single-stranded DNA rather than RNA or double-stranded DNA (1). Sigal and his colleagues adopted basic techniques such as electron microscopy, DNA denaturation, and polymerase assay using [3H]TTP (2*105 cpm/nmole) for measurements of DNA synthesis (1). E. coli polymerase II (fraction V, 270 units/mg), T4 gene-32 protein, E. coli polymerase III (10,000 units/mg), E. coli exonuclease III (180,000 units/mg), and DNA polymerase I (180,000 units/mg) were prepared for the experiment in order to show SSB stimulates the DNA synthesis by these polymerases (1). Used materials and methods are as follows (1):

     a. DNA from bacteriophages lambda and T7 was used by extracting the purified phage with phenol.
     b. DNA template was treated by exonucleas III (incubation of 1mmol of DNA with 2500 units of exonuclease III for 5 minutes at 30ƒC in 4.5 ml of a buffer containing 0.09 M Tris-acetate (pH 8.2)-2 mM 2-mercaptoethanol-2 mM MgCl2).
     c. The reaction is terminated by heating for 5 minutes at 65ƒC.

In this experiment, they concluded that the DNA-unwinding protein, which is called SSB later, binds tightly to single-stranded DNA by eluting single-stranded DNA cellulose with a solution of 0.9 mg/ml of dextran sulfate and by using standard fractionation techniques (1). After Na dodecyl SO4-polyacrlamide gel analysis and electron microscopy, only 22,000-dalton protein was observed to form complexes with SSB (1). Sigal and his colleagues also determined the form of SSB with circular, single-stranded fd DNA by electron microscopy. They showed a "beaded necklace" complex that has 45-50? diameter and "beads" spaced at about 60? intervals along the extended DNA single-strand (1).

- Function and characteristics
Single-stranded DNA-binding proteins (SSB) have high affinity to single-stranded (ss) DNA and participate in DNA replication, recombination, and repair as accessory protein (2). SSB plays a role in separating DNA strand during replication and prevent ssDNA from re-form a double helix. The monomer contains 177 amino acids with an isoelectric point of 6.0 (2). Without DNA, it exists as a homotetramer that is stable at 30nM at 25ƒC (2). According to Lohman and Ferrari, the binding site of Eco SSB is contained within the first 115 N-terminal amino acids (2). According to Chase and Williams, SSB can bind four d(pT)8, two d(pT)16, and one d(dT)30-40 molecule based on sedimentation experiments (3). Each SSB monomer can interact with 6-8 basee (3). There are two kinds of complexes of SSB-ssDNA in different site sizes in the (SSB)35- and (SSB)65- binding modes. Single-stranded DNA can interact with two SSB subunits in the (SSB)35 complex, which has "smooth-contoured" structure as well as with all four SSB subunits in the (SSB)65 complex, which has "beaded" structure (2,3). SSB binds to ssDNA with two different types of intertetramer cooperativity: "Unlimited" cooperativity and "Limited" cooperativity (2). In "Unlimited" cooperativity, long protein clusters can be formed because the nearest neighbors interact each other on both sides of a bound protein. This type seems to be associated with the (SSB)35- binding mode. So, a single protein cluster saturates the ssDNA (2). On the other hand, in "Limited" cooperativity, which is related to the (SSB)65- binding mode, protein clusters are limited to the formation of dimmers of tetramers because of the interaction of four SSB subunits with ssDNA (2).
 

Reference
(1) Sigal, N., Delius, H., Kornberg, T., Gefter, M. L., Alberts, B. M. 1972. Proc. Nat. Acad. Sci. USA 69:3537-41
(2) Lohman, T. M. and Ferrari, M. E. 1994. Annu. Rev. Biochem. 63:527-70.
(3) Chase, J. W. and Williams, K. R. 1986. Annu. Rev. Biochem. 55:103-133.