The Mechanism of Replication

1. E. coli DNA synthesis genes. The overall process of replication requires the products of genes dnaA, dnaB, dnaC-D, dnaE and dnaG. The products of genes dnaA and dnaC-D are required for initiating new rounds of replication and the products of dnaE and dnaG for replication fork movement. The dnaG protein appears to be necessary for initiation of synthesis of DNA fragments.

2. Replication starts with an incision. The DNA of chromosome consists of many replication units or replicons. According to one model replication is initiated in each replicon only after a break in one of the two parental strands.



3. Origin. Replication starts at a unique site (called the origin) on the chromosome. In E.coli the origin of replication is at about 74minutes on the chromosome map.

4. Role of membrane. It has been suggested that the replicating apparatus in E.coli consists. of an enzymatic complex at the point of attachment of the DNA to the plasma membrane.



5. Unwinding of the strand. The DNA double helix has to unwind in order to separate the two strands for replication. The unwinding of the DNA double helix is brought about the DNA unwinding proteins. The proteins bind preferentially to single strands of DNA and promote unwinding of double helical DNA.



6. Superhelix relaxing protein (swiveling protein). As the replication fork moves down the DNA double helix the parental strands winds. The extent to untwisting is one turn for every 10nucleotides laid down.

7. RNA Primer. Initiation of DNA synthesis requires an RNA primer. DNA polymerases cannot initiate DNA chains de novo. (RNA polymerases, cannot initiate DNA chains de novo. (RNA polymerases, in contrast, can intiate chains de novo during transcription). A per-existing polynucleotide chain (the primer) is required to which polynucleotides are added.



8. Chain elongation. Synthesis of the new DNA strand takes place by addition of DNA nucleotides to the 3’ – OH group of the last ribonucleotide of the RNA primer. The newly made is joined by DNA ligase to form high molecular weight DNA (site A). RNA primers which are joined on both sides to DNA chains are digested by specific endonucleases, e.g. ribonuclease H.


9. Replication has direction. Replication may proceed in one or both directions from the point of origin. Replication in one direction is called unidirectional replication, while that is both direction is called bidirectional replication.



10. Replicating forks. At a point where the two strands are separated a replicating fork is formed. The fork appears in the form of a Y and advances during replication. In bidirectional replication the separateds between the two forks appear as a ‘bubble’ or an ‘eye’ under the electron microscope. The speed or extent of movement of the two forks in opposite directions need not be the same.

11. Okazaki fragments. The duplication of the DNA strand is brought about by the movement of the replication forks. Replication of the two parental strands takes place simultaneously as the fork move along.
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12. Polynucleotide ligase. As mentioned previously, replication is a discontinuous process and takes place in short segments called Okazaki fragments. Polynucleotide ligase joins these fragments into high molecular weight DNA. In mutants which produce defective ligase, the joining of Okazaki fragments is greatly impaired.