Molecular Genetics

Initiation of DNA replication at origins leads to the concept of a replicon.

Facts

• A replicon is all DNA synthesized from the same origin of replication.
• For simple replicons the position of the origin in a replicon can be determined by restriction prior to electron microscopy.
• For DNAs with multiple replicons, the rate and direction of fork movement can be visualized by pulse-chase experiments and DNA fiber autoradiography).

In DNA fiber autoradiographs produced from synchronously dividing cells, a series of similar patterns are found on the same fiber. Other fibers may have other patterns. Similar results were obtained by electron microscopy.

Interpretations

• The origin of replication in a replication bubble will be in the middle of the bubble if replication is uniformly bidirectional, at one fork if replication is unidirectional, or closer to one end than the other if the average rate of fork movement differs for the two directions.
• In the circle example, replication is principally unidirectional with the origin a short distance clockwise of the EcoRI site.
• In the fiber example, the symmetric pattern centered around the replicated fibers suggests equal bidirectional fork movement.
• That adjacent replicons in DNA fibers appear equivalent in size suggests that their replication was initiated synchronously.
• For chromosomes with a single origin of replication (SV40, polyoma, mitochondria, many bacteria), the replicon is identical to the chromosome.

Further information

• Different replicon clusters may initiate replication at different times. Clusters that replicate at the same time are said to belong to the same replicon family.
• In a more modern variation of DNA fiber autoradiography, two different tagged dNTP precursors are used sequentially for the pulse and chase periods. They are detected by fluorescent antibody staining. In addition, specfiic DNA sequences can be identified by FISH. Performing these analyses on molecularly combed fibers allows alignment and comparisons of the same DNA sections from different cells. The technique played roles in demonstrating that firing of an origin in mammalian cells inhibits firing of origins in the vicinity and in resolving a controversy over the possible existence of diffuse origins (ref).