Overview of E. coli single stranded binding protein SSB and its cooperativity.

E. coli single stranded binding protein (Eco SSB) is encoded by ssb gene which consists of 534 bp in coding region. Product of ssb gene contains 177 amino acids with a molecular weight of 18,9 kDa. In solution, Eco SSB forms a tetramer as the active form (sometimes be viewed as a “dimer of dimers”). SSB is known to tightly bind single stranded DNA (ssDNA) with very high affinity and protects that ssDNA from nuclease, reannealing or secondary formation …  or keeps ssDNA intermediates, therefore involved Continue Reading →

What is DNA polymerase switch?

DNA polymerase switch is an activity whereby a polymerase is replaced by the other polymerase. There are 5 distinct DNA polymerases in the E. coli cell: Pol I, Pol II, Pol III, Pol IV, Pol V, each has different roles in genomic maintenance and evolution. Among these 5 polymerases, DNA Pol III Holoenzyme (Pol III HE) is a primary enzyme for DNA replication that can synthesize new strand with very high processivity (more than 50 kb per binding event) and high speed (about 1kb per second). However, Pol III HE is Continue Reading →

Why DNA Polymerase III Holoenzyme can synthesize DNA with very high fidelity?

As you well know, DNA Polymerase III Holoenzyme (Pol III HE) is the major polymerase involved in DNA replication. During DNA replication, new strand is synthesized by adding nucleotides to the 3′ terminus of the primer that are complementary to the original DNA template strand. DNA synthesis by Pol III HE is very high fidelity with the error rate is low as 10–6 to 10–8. So, which factor contributes to this accuracy of Pol III HE? Pol III HE is a big molecule that consists of 10 different subunits. Continue Reading →

Why DNA gyrase is needed during DNA replication?

During DNA replication, double stranded parental DNA need to be separated by helicase to produce two single stranded DNA which are used as as template (leading and lagging template) for DNA synthesis by DNA polymerase. However, this unwinding activity by helicase accumulates a number of positive supertwisting in front of replication fork. A saturated level of this positive superstwisting will inhibit further elongation because helicase is unable to unwind the positive supercoil-template. To solve this problem, in E. coli cell, a topoisomerase II known as gyrase, will work to remove this stress Continue Reading →

What is Klenow fragment?

E. coli Polymerase I is a product of polA gene that consists of 5′-3′ polymerase activity and both 3′-5′ and 5′-3′ exonuclease activity. In vivo, Pol I takes part in DNA synthesis and DNA repair. The 5′-3′ exonuclease activity of Pol I makes it unsuitable for many applications in vitro. However, it is found that a protease known as subtilisin, can cleave Pol I into a small fragment, which contains the 5′ -> 3′ exonuclease activity, and a large fragment that consists of both 5′-3′ polymerase activity and 3′-5′ exonuclease activity called Klenow Continue Reading →

« 1 2 3 4 »