1 Proofreading of newly synthesized DNADr. MAHA SMAISM

2 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

3 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.

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6 Solving the problem of supercoils

7 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.

8 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.

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10 To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix 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 .

11 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.

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