proofreading

Proofreading of newly synthesized DNA

Dr. MAHA SMAISM

It is highly important for

the survival of an organism that the nucleotide sequence of DNA
be replicated with few errors as possible. Misreading of the template sequence could result in mutations.
To ensure replication accuracy, DNA polymerase III has, two proofreading enzymes:
3'→5' exonuclease 5‘ to 3' DNA polymerase activity,and “proofreading” activity

As each nucleotide is added to

the chain, DNA polymerase III checks to make certain the added
nucleotide is correctly matched to its complementary base
on the template. If it is not, the 3‘ to 5' exonuclease activity corrects
the mistake.

For example, if the template

base is cytosine and the enzyme mistakenly put an adenine
instead of a guanine into the new chain, the 3'→5' exonuclease
activity removes the misplaced nucleotide. The 5' →3'
polymerase activity then replaces it with the correct nucleotide
containing guanine [Note: The proofreading
exonuclease activity requires movement in the 3'→5' direction, not
5' →3' like the polymerase activity.

Solving the problem of supercoils

As the two strands of the double
helix are separated, a problem is occurred, the
appearance of positive supercoils (also called supertwists) in the
region of DNA ahead of the replication fork.

Supercoiling can be demonstrated by

tightly grasping one end of a helical telephone cord while twisting
the other end. If the cord is twisted in the direction of tightening
the coils, the cord will wrap around itself in space to form positive
supercoils.
If the cord is twisted in the direction of loosening the
coils, the cord will wrap around itself in the opposite direction to
form negative supercoils.

Type I DNA topoisomerases: These enzymes reversibly cut

one strand of the double helix. They have both nuclease
(strand-cutting) and ligase (strand-resealing) activities. They
do not require ATP, but rather appear to store the energy from
the phosphodiester bond they cleave, and reusing the energy to
reseal the strand .
To solve this problem, there is a groupof enzymes called DNA topoisomerases, which are responsiblefor removing supercoils in the helix

Type II DNA topoisomerases: These enzymes bind tightly to

the DNA double helix and make temporary breaks in both
strands. The enzyme then causes a second stretch of the DNA
double helix to pass through the break and, finally, reseals the
break . As a result, both negative and positive
supercoils can be relieved by this ATP-requiring process.