Question

Describe the synthesis of an Okazaki fragment on the lagging strand in a replication fork. Include names of enzymes and the proper 5’ and 3’ ends of DNA strands.

Answer 1

SOLUTION:

Replication starts from the origin of replication to the terminus and is accompanied by the movement of the replication point, called the Replication fork.

• REPLICATION FORK:

Transient structure generate during replication.

It can be of 2 types:

1. Unidirectional (Rare)
2. Bidirectional (Common)

• UNIDIRECTIONAL(Rare):

1 Replication fork

Found in some plasmid only.

• BIDIRECTIONAL(Common):

2 Replication fork.

1. Unidirectional Replication:

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• DNA Polymerisation (5'-3')
• Unwinding by Helicase (5'-3')

Rule: Helicase always binds with template of lagging strands.

1. At each growing replication fork, one strand called the leading strand,is synthesized continously from a single primer on the leading strand template and grows in the 5'-3' direction.

The growth of the leading strand proceeds in the same direction as the movement of the replication.

2. Synthesis of the lagging strand is more complicated because DNA polymerases can add nucleotides only to the 3' end of a primer or growing DNA strand.

The leading strand synthesis can start as soon as dsDNA is denatured and template is exposed but the synthesis of the lagging strand must wait for movement of the replication fork to exposed but the synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of template before it can be replicated.

After 1000-2000 nucleotides of the leading strand have been replicated, the first round of lagging strand synthesis can begin.

As compare to leading strand, it is synthesized discontinously from multiple primers. Short pieces of DNA , called OKAZAKI FRAGEMENT, are repeatedly synthesised on the lagging strand template.

FIGURE SHOWING MECHANISM OF BIDIRECTIONAL REPLICATION

Synthesis of leading and lagging strands of DNA.​​​​​​​

DNA helicase, DnaB , separates the two strands of the double helix. It bind to the lagging-strand template at the replication fork. DNA helicase move along ssDNA in a defined direction. This property is referred to as the polarity of the DNA helicase . The DnaB at the repplication fork travel along the lagging-strand template in a 5'-3' direction.

DNA primase,DnaG, associated with the DNA helicase and sythesizes RNA primers. DNA primase make transient interaction with helicase. It periodically associates with DNA helicase, synthesizes a new RNA primer and dissociates . The relatoively weak interaction with DNA primase and DNA helicase regulates the length of OKAZAKI FRAGEMENTS.

A tighter association would result in a more frequent primer synthesis on the lagging strand and therefore shorter okazaki fragements. Similarly, a weaker interaction would result in a longer okazaki fragements.

FIGURE SHOW ENZYMES NAME:

These enzymes play an important role in synthesis of okazaki fragement in the replication fork.