Molecular Genetics

Facts

	Recall several facts about DNA and its replication: DNA polymerization occurs in the 5' to the 3' direction on the synthesizing strand by the joining of the 3' oxygen of the nascent strand to the a phosphorus of the incoming dNTP. Almost all DNA polymerases can only elongate existing DNA chains. They can not initiate chains. In contrast, RNA polymerases can start polynucleotide chains. The double-stranded DNA has its strands in anti-paralell orientation. Melting of the double helix is coupled to polynucleotide polymerization. The above considerations lead to a picture of the replication fork diagrammed at left. Note the directions of strand synthesis relative to the direction of fork movement.
The results of a pulse-chase labeling experiment of replicating DNA are shown at right. Sedimentation was used to separate single strands based on their size. Note the presence of short strands (Okazaki fragments) after the pulse in addition to long DNAs and note that these fragments disappear on chasing the label (darker line).

Interpretations

• That the direction of synthesis of one of the new strands is opposite to that of the fork requires that strand to be made discontinuously. It is called the lagging strand.
• The other strand is continuously synthesized. It is designated the leading strand.

Further information

• Lagging strands are initiated from short RNAs synthesized by a "primase" activity. The RNA is subsequently removed and the gap filled in and ligated to the next segment.
• In plastid DNA replication, RNA removal is inefficient, resulting in the plastid genome being an RNA-DNA chimera.
• In E.coli, leading and lagging strand synthesis is accomplished by a pseudodimeric DNA polymerase enzyme, one half of which works on the leading strand and the other on the lagging strand.
• Several proteins act at the replication fork.
• In eukaryotes two separate enzymes are used. The a enzyme has a primase activity and initiates lagging strand synthesis. The g enzyme has no primase activity and elongates the leading strand. It complexes with PCNA protein which increases enzyme processivity by clamping around the DNA, doughnut-like.
• One hypothesis to account for the universal 5' to 3' polymerization direction is that 3' hydroxyls are much more stable than 5' triphosphates.
• A shockwave animation of the replication process is available.

This is page 1312 of Molecular Genetics by Ulrich Melcher, © 1998, 1999, 2003