4. Dna Polymerase

There are three enzymes, termed DNA polymerases, that are used to replicate DNA. Polymerase III copies the strand while I and II are responsible for proofreading and repairing the copied strand. These polymerase enzymes catalyze the addition of nucleotides to elongate the newly forming strand using the parent strand as a complementary template. A short chain of nucleotides, called a primer, is required by the polymerase to start. From this primer the rest of the strand can be formed. The enzymes can only add nucleotides in 5` to a 3` direction beginning with the 3` end of primer. The DNA polymerase catalyzes the nucleophilic attack of the 3`-OH from the growing strand on the α-phosphate of the nucleotide, releasing pyrophosphate. Thus, the polymerases can only add nucleotides.

5. Okazaki Fragments

Each strand of duplex DNA runs in an anti-parallel (opposite) direction to the other. One problem the replication machinery has to overcome is that the polymerase only travels in the 5` to 3` direction, yet must always move towards the replication fork. To solve this dilemma, the polymerase builds the lagging strand using many pieces, called Okazaki fragments, in a leapfrog fashion from one primer to the next. The leading strand simply follows in the direction of the replication fork.

6. Steps of dna replication

1. Unwinding the helix

The first step in DNA replication is the formation of the replication fork. The enzyme gyrase induces supercoiling to unwind the double helix. Following the gyrase is helicase. Helicase breaks the hydrogen bonds between the base pairs, allowing the parent strands to separate. The helicase obtains the energy for this process by the hydrolysis of adenosine triphosphate (ATP).

2. Stabilizing the Strands

The phosphodiesteric bonds of the single-stranded DNA are now vulnerable to attack through hydrolytic cleavage. Single-stranded DNA binding (SSB) proteins prevent these bonds being broken and keep the separated strands from reannealing the replication process.

3. Primer addition

The enzyme primase creates an RNA primer from which nucleotide polymerization can begin. The lagging strand has the additional complexity of the Okazaki fragments because of the 5` to 3` polymerization requirement and also because overall polymerization must move towards the replication fork.

4. Nucleotide Addition

After the attachment of RNA primer, DNA polymerase III adds nucleotides to the new strand in a 5` to 3` direction. Because the polymerization on both strands must move towards the replication fork, the lagging strand replication is more complex, requiring the use of Okazaki fragments.

5. Primer Removal

The RNA primers are then removed by DNA polymerase I, which also fills in the resulting gaps with DNA. The polymerase is unable to connect the 3` nucleotide of the replacement nucleotides to the rest of the strand, leaving a phosphodiesteric gap in the strand.

6. Filling the Gaps

Lastly, the nicks between the Okazaki fragments on the lagging strand must be joined. This is accomplished by the enzyme ligase. This reaction requires the breakup of one ATP molecule into AMP and PPi.