question on T4 polymerase

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catalogi
catalogi's picture
question on T4 polymerase

Dear,

I have a question on the T4 polymerase
I found the following phrase in a textbook:

T4 polymerase will either elongate or remove nucleotides and this depends on avaibility.

What does this mean?

Example lets say we have
5' GTACGGAT 3'
3' CATGCCTACGTAC 5'
and we add T4 polymerase and some dNTP's , what will happen then?

I suppose the T4 polymerase will add thise nucleotides ? untill the template is completed?

But what do they then mean with "remove nucleotide" ?

anyone an idea?

The FFM
The FFM's picture
In the presence of

In the presence of nucleotides T4 will fill in 5' overhangs to blunt the fragment by adding nucleotides to fill in the overhang.

In the absence of available free nucleotides, T4 ploymerase will remove the 3' overhang to blunt it.  T4 removes the overhang by digesting away the additional overhanging nucleotides (i.e it acts as an "exonuclease"). The 3' to 5' exonuclease activity of T4 is much greater than for an equimolar amount of DNA pol I. 

In the example you provided, as long as you include nucleotides, T4 will blunt the fragment by filling in the 5' overhang.  If no nucleotides are provided T4 will do nothing. 

If you need an enzyme with 5' - 3' exonuclease activity to "chew back" a 5' overhang use Mung bean exonuclease.

Quick Reference
http://www.neb.com/nebecomm/products/faqproductM0210.asp#123

catalogi
catalogi's picture
thanks a lot.

thanks a lot.

But what happens when you added a liltle amount of dNTP's or when you added onlu G and C's... what then?
Will the T4 then add the G and C's and then simply start removing the 3' overhang (if there is one, in this case there isnt one, but lets say whe change the template to:
5'       ACGGAT              3'
3' CATGCCTACGTAC 5'