DNA Replication -Enzymes

1.DNA polymerase I (kornberg enzyme)
DNA DEPENDENT DNA POLYMERASE (BY ARTHUR KORNBERG )

• It makes an average of 20 phosphodiester bonds before dissociating from the template.
• Helps in proof reading.
• DNA polymerase I has nucleolytic (depolymerizing)activities, which are an intimate part of their function. The 5′ to 3′ exonuclease activity removes base‐paired sequences ahead of the 5′to3′polymerizing activity. During replication, this can remove primers .
• DNA polymerase I (and of the other forms of DNA polymerase found in E. coli) is the 3′ to 5′ exonuclease activity. This activity can de‐polymerize DNA starting from the newly synthesized end.
• The 3′ to 5′ exonuclease activity serves an editing function to ensure the fidelity of replication. It helps in repair mechanism .

1 .Among the following which activity is absent in bacterial DNA polymerase- I   (2008 CSIR DEC )

a. 5'-3' polymerase activity                      c.5'-3' exonuclease activity

b.3'-5' polymerase activity                       d.3'-5'exonuclease activity 

                                 2. DNA polymerase -II

DNA polymerase II is a specialized repair enzyme. The enzyme is more processive than Pol I. Pol II has the same editing (3′ to 5′) activity as Pol I, but not the 5′ to 3′ exonuclease activity. It is known to restart the replication process when a fork stalls at a time of damaged Dna.
                                                   
            3.DNA polymerase-III (Replicase) A heterodimer composed of 10 different subunits . Having high polymerization activity (10^5 nut/ min.)It is the true enzyme responsible for elongation process .

                            Image source credit - http://www.nature.com/nrm/journal/v3/n5/images/nrm804-f4.jpg

• It is the actual replication enzyme in E. coli . 
•  Pol III is much more processive than the other enzymes, making about 500,000 phosphodiester bonds on the average.
•  It lacks a 5′ to 3′ exonucleolytic activity, although a subunit of the enzyme carries out the editing (3′ to 5′) function during replication. It carries 3'-5' exonuclease activity (Epsilon unit ) and  Theta subunit assist the epsilon sub unit plays an important role to ensure the replication fidelity.
• It carries 5'-3' polymerase activity by alpha subunit .
•  Pol III synthesizes DNA at least a hundred times more rapidly than the other polymerases. It can synthesize half of the bacterial chromosome in a little more than 20 mins. 
• It consist of 3 complexes -a. y - complex (loads the clamp in the template ) b.core enzyme (alpha subunit ,epsilon subunit,theta subunit )c.Beta clamp (2 beta sub units). 
• Tau subunit -Helps in dimerization .

Dna polymerase IV and V (Translesion DNA polymerases )

• 4.Topoisomerase -I it creates single stranaded nick, need no ATP, change linking no. by 1.         
•   5.Dna gyrase(Topoisomerase-II)-A swivel to relieve positive supercoiling in advance of the replication fork. It needs ATP, create double stranded nick, change linking no. by 2 . It is needed to decatanate the two circular ds Dna daughter molecules at the end of the replication .
• Inhibitors-  Nalidixic Acid and Ciproflaxin inhibit topoisomerase activity. Novobiocin inhibits binding of ATP to Topoisomerase II.

• DNA ligase- Seals off the nicks by using energy from  phosphodiester bonds in the form of  ATP or NAD to join a free 3′ hydroxyl with an adjacent 5′ phosphate. 
• Helicase(Dna-B)-It is responsible for unwinding of the ds DNA by disrupting the H-bonds between the (5'-3' and 3'-5' )antiparallel strand .

Telomerase- Synthesis of DNA  at ends of chromosome.
Eukaryotic Dna polymerase alpha has tightly associated primase activity but moderate processivity followed by polymerase switching with E and delta .
Dna polymerase E and delta are highly processive but lack primase activity .

 
Q1.Which DNA polymerase lacks proof reading activity ?
Ans. DNA Polymerase α (It initiates replication and synthesize primers -Dna G)
Q2. Active site of topoisomerase I  contains Tyrosine . (IMP.)
3.Replicative Dna polymerases are Dna pol gamma and Dna pol epsilon .
4.PCNA -Proliferating cell nuclear antigen (It confers high processivity ) It is a homodimer . It is eukaryotic counterpart of the y subunit sliding clamp of E.coli.
5.RPA (Replicative protein A ) Its a ss binding protein and helps in unwinding of the helix.Also called as eukaryotic counter part of SSB protein of E.coli.
6.RFC -(Replication factor C) It is the  eukaryotic counterpart of the complex clamp loader of E.coli.

NOTE -Topoisomerases can cut and re knot the DNA strands. They break the phosphodiester bond between 2 adjacent bases. Surprisingly they dont use ATP hydrolysis energy to cleave the high energy phosphate bond because topoisomerases form phospho tyrisine bond which conserves the energy that is used to form the ester bond to rejoin the DNA.

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