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Wednesday, November 4, 2015

Klenow Fragment - Sturctural Analysis, Comaprison and Applications

What is Klenow Fragment??

  • Klenow Fragment is the proteolytic cleavage product of DNA Polymerase I. 
  • Klenow Fragment is generated when DNA polymerase I is cleaved using protease subtilisin. 
  • Klenow fragment retains 5’-3’ polymerase activity and 3’-5’ proof reading activity. 

Structure of DNA Polymerase 

DNA Polymerase has three domains:

1. Polymerase domain.
2. Proofreading domain.
3. Exonuclease domain.

Klenow Fragment has only 2 domains:

1. Polymerase domain 
2. Proofreading domain

Klenow Fragment lacks 5'-3' exonuclease domain.

Structural Comparison of DNA polymerase and Klenow Fragment

Applications / Advantages of Klenow Fragment
  • DNA sequencing by the Sanger dideoxy method.
  • Fill-in of 5´ overhangs to form blunt ends.
  • Removal of 3´ overhangs to form blunt ends.
  • Second strand cDNA synthesis.
  • Second strand synthesis in mutagenesis protocols.

NEB Technical Resources
Technical Resources, PDB
Other Internet Sources 

Saturday, May 16, 2015

Base Excision Repair Mechanism

Base Excision Repair Mechanism, is a DNA repair mechanism, where in the altered bases of the DNA, is removed or replaced. In Base Excision repair, glycosylase recognizes and removes the damaged base by hydrolysing the glycosidic bond.

The resulting abasic sugar, is removed from the backbone by endonucleolytic action.
Endonucleolytic cleavage also removes a-purinc or a-pyrimidinic sugars that arise by spontaneous hydrolysis.
After the removal of damaged nucleotide, DNA polymerase and DNA ligase act upon the damaged strand to restore.

Cells have different DNA glycosylases, with different specificities. For example Uracil glycosylase which specifically removes the damaged uracil.

Lets see How Does Glycosylase Detects damaged base?

Glycosylases, diffuse through the minor groove of the DNA until a specific kind of lesion is detected. Glycosylases removes, the damaged base by a mechanism called Base Flipping.
The damaged base project out or flip out of the DNA double helix, glycosylase goes and binds to that base and removes it.

Glycosylase can selectively cleave the nucleotide without cleaving the DNA backbone. When the nucleotide is removed, a gap is created. To repair the strand with the gap, the strand need to be cut out and that short stretch removed need to be synthesized. The site at which the gap is present is called as the AP site. apurinic site or the apyrimidinic site .

Once the gap is recognized on the DNA strand, AP Endonuclease comes into the scene to nick the strand. This is because for the DNA Polymerase one to act on the strand, DNA need to be nicked then only DNA polymerase start its function, that’s the reason AP endonuclease acts on the damaged strand to create a nick.

DNA Polymerase has two activities, exonuclease activity and the polymerase activity. In this case, DNA polymerase makes use of three prime to five prime exonuclease activity, to chew off some the bases, and at the same time DNA polymerase, starts adding up the chewed off bases by its polymerase activity.

Once polymerase completes its action Ligase, seals the nick and completes the process.

Sunday, May 10, 2015

p53 Structure and Function - Cell Cycle Regualtion & Apoptosis

P53 is a tumor suppressor gene, present in eukaryotic cell. Protein encoded by p53 regulates cell cycle and function as tumor suppression, hence it is also known as TP53 or tumor protein.

p53 is referred to as “the guardian of the genome”, as its role in conserving stability by preventing genome mutation. “53” denotes molecular mass of p53 as it runs as 53KDa band on SDS – PAGE.