DNA Replication

Although much of our focus will be on the replication process in E. coli, there are some general features which are shared by all replication systems and which you should be familiar with:

• Semi-conservative: All DNA replication is semi-conservative. This means that the product of replication is composed of a newly synthesized strand of DNA that hydrogen bonded to an original (template) strand.


• DNA Polymerase: DNA replication is carried out by an enzyme that is a type of DNA polymerase. Most cells have multiple DNA polymerases that carry out replication under specific conditions, such as chromosome replication or DNA repair. The DNA polymerase that performs specific functions will be mentioned when that function is described. All DNA polymerases catalyze the formation of a phosphodiester bond between the 3' OH of the new strand being extended and the 5' P of a free dNTP (the base depends on what base exists on the template at that position). Therefore, the new strand always grows in the 5' to 3' direction.


• Primer: The polymerization described above occurs between an existing 3' OH and an incoming dNTP. DNA polymerases cannot incorporate a dNTP without adding it to the 3' OH of a nucleotide that is part of a double-stranded molecule. Therefore, DNA replication requires a primer which is a short nucleic acid that is bound to the template and which has a 3' OH for DNA polymerase to utilize. RNA polymerases are capable of adding a dNTP de novo, so replication always begins with an RNA polymerase - in this case referred to as a Primase - that generates a short (6 - 8 bases) RNA fragment that is complementary to the template. This "primer" can then be extended by a DNA Polymerase. The Primase is NOT the same as the RNA polymerase that is involved in Transcription.


• Lagging strand: Since DNA polymerases only synthesize new strands in the 5' to 3' direction, the movement of a growing fork along the chromosome presents a problem: Only one of the new strands is "growing" in the same direction as the fork is moving. The other strand needs to be replicated in the opposite direction. This strand is defined as the lagging strand. The strand growing in the same direction as the fork movement is the leading strand.
  

  The cells solves this problem by replicating the lagging strand discontinuously. This means that it replicates short segments (a few thousand nucleotides) of the lagging strand "against the grain" of the fork movement. These short fragments can then be joined together to form a full DNA strand. Basically, as the growing fork moves along, strand separation reveals the templates. Once a few thousand nucleotides of the lagging strand template has been exposed by strand separation, a primer is synthesized and this is then extended by DNA polymerase in the direction against the movement of the fork. This short fragment is referred to as an Okazaki fragment. By the time DNA polymerase reaches the end of the previous Okazaki fragment, the growing fork has moved further along revealing more of the lagging strand template and the process can be repeated. This is nicely illustrated at this link.

  The page on Elongation will deal with how Okazaki fragments are pieced together into one long molecule.

• Supercoiling: Supercoiling of DNA is covered here. All replication systems have topoisomerases involved that deal with the positive supercoiling that results from the movement of the growing fork.

This video demonstrates this process of discontinuous synthesis of the lagging strand: