Molecular Genetics

Facts

The polymerization of DNA that is central to the replication of chromosomal DNA occurs at replication forks.

• Recall that DNA replication is semi-conservative. Semi-conservative replication requires melting of the double-helix prior to polymerization of the daughter strands. Whether melting is independent of polymerization or whether the two processes are coupled can not be concluded from the Meselson-Stahl experiment.
• Cairns used the electron microsocope to observe small circular double-stranded DNA molecules that had been replicating in E. coli cells.
• He observed structures such as those diagrammed at right. The structures contained eyes (or bubbles). The thickness of the strands were the same whether they were in the main circle or the eyes.
• Analysis of a population of replicating DNAs revealed a variety of bubble sizes. Both sides of the bubble had equal contour lengths.
• The junction between the eye and the main circle is the replication fork.
• Note that the eye segments are not extensively interwound.

Interpretations

• The replication bubble enlarges as replication proceeds.
• The double-stranded DNA template is not completely denatured before DNA synthesis begins. Rather, unwinding and synthesis are coupled. This is indicated by the uniformly equal thicknesses of the bubble walls.
• The DNA template at the replication fork must be unwound to allow access by the DNA polymerase and DNA synthesis.
• An enzyme activity is needed to relieve superhelical stress that results from the unwinding process. Otherwise, the newly replicated bubble would be extensively interwound.

Further information

• Enzyme activities that unwind double-stranded nucleic acids are called helicases.
• Enzymes that relieve superhelical stress change the linking number of the DNA and are called topoisomerases.
• Unwinding and synthesis appear also to be coupled in most other systems studied.
• One exception is in the replication of mitochondrial and plastid DNA. A new strand is synthesized on a segment of one of the template strands, but the other strand is not copied til later. This results in a single-stranded displacement loop.
• The synthesis of rDNA may also proceed through displacement loops (ref).

This is page 1311 of Molecular Genetics by Ulrich Melcher, © 1998, 1999