DNA Replication

DNA replication is a complex process of synthesis of DNA molecules. This process occurs in the S phase of the cell cycle. Each DNA molecule forms two new daughter DNA molecules.

 Models of DNA replication

Three possible patterns of the DNA replication process are proposed. The actual DNA replication mode is called the semiconservative model. The two others are theoretical.  

a.     Semiconservative DNA replication

According to this model, the parental DNA molecule helix unwinds and unzips, and both the strands acting as a template synthesize the new daughter DNA. The new strands are synthesized complementary to both old strands. If T (thymidylic acid) is present, A (adenylic acid) is added; if G (guanidylic acid) is present, C (cytidylic acid) is added; likewise, A would attract T, and C would attract G. Each replicated DNA molecule would consist of one “old” and one “new” strand, hence the reason for the name semiconservative replication.  Watson and Crick proposed this model for DNA replication. They proposed that DNA replication is semiconservative.

b.     Conservative DNA replication

This theoretical mode of replication also relies on the parental strands as a template. According to this model, the complementary polynucleotide chains are synthesized. Following synthesis, however, the two newly created strands then come together, and the parental strands also recombine. The original DNA helix is thus “conserved.”

c.      Dispersive DNA replication

According to this theoretical mode, the replication of new DNA also relies on the parental strands as a template. In this model, the parental strands are dispersed into two new double helices following replication. Hence, each strand consists of both old and new DNA. This mode would involve cleavage of the parental strands during replication.

Meselson-Stahl experiment- Replication as a semi-conservative process

Matthew Meselson and Franklin Stahl in 1958, provided strong evidence that semiconservative replication is the actual mode used by cells to produce new DNA molecules. They experimented on E. coli cells in a medium that had ¹⁵NH₄Cl (ammonium chloride) as a source of heavy nitrogen containing one more neutron than the naturally occurring ¹⁴N isotope.

Mathew Meselson and Franklin grew bacteria with ¹⁴N in their DNA in a growth medium with a heavy isotope of ¹⁵N nitrogen for many generations. After many generations, almost all the bacteria had heavier isotope ¹⁵N in their DNA. The newly formed bacteria were shifted to a ¹⁴N medium containing only ¹⁴NH₄Cl. These cell samples are then removed back from the medium and centrifuged. Three separate samples were obtained. One sample was obtained just after the cells were introduced into the ¹⁴N medium. This sample was named 0 sample. The second sample was obtained just after 20 minutes, called sample 20. The other sample was obtained after another 20 minutes, called sample 40 minutes.

DNA samples were obtained from all the bacteria and were dissolved into cesium chloride and then spun at a very high speed in an ultra-centrifuge for many hours. The DNA of normal bacteria appeared the lightest as formed sediment at the top of the test tube, while the DNA of sample 0 minute appeared heaviest as it formed sediment at the bottom of the test tube. The DNA sample at 20 minutes formed sediment intermediate level to that of the natural sample and 0-minute sample. The sample 40 minutes had two sediments, one at the top and the other at the intermediate level.

Meselson and Stahl interpreted their results as follows; The DNA of the control sample had both the strands of ¹⁴N, whereas the DNA sample of 0 minutes had both the strands ¹⁵N and the DNA of the sample 20 minutes had one strand of ¹⁴N, and the other of ¹⁵N.

 

Process of DNA replication

The process of replication involves the following main steps

            a.      Uncoiling of DNA helix

DNA has a helical structure. Before replication, Topoisomerase, also called DNA gyrase, makes a single-strand cut and hydrolyzes the phosphodiester linkage. It causes the uncoiling of the double helix.

b.     Unwinding of the duplex

After uncoiling helicase breaks the hydrogen bond between the nitrogen bases forming the replication bubble. The ends of the replication bubble are called replication fork.

 c.      Assembly of SSB proteins

Both strands tend to reunite forming a duplex. Therefore, to prevent the formation of the duplex SSB proteins bind to 8-10 nucleotides on a single strand of the two strands.

d.     Assembly of primer

The key enzyme of the replication process is the DNA polymerase III. This enzyme cannot add the 1^st nucleotide and requires a preexisting nucleotide in the new DNA strand. This is provided by a primase enzyme which synthesizes an RNA sequence of about 10-30 basses RNA primer. The primer is a small segment that provides the OH end for the next nucleotide.

 e.      Elongation/Polymerization

The DNA polymerase-III adds complementary nucleotides to both the template strands. The DNA polymerase remains in the replication fork on the template strand and continuously adds nucleotides in 5-3 directions to the new strand as the fork progresses. The two strands of a double helix are antiparallel to each other, one runs in the 5′ to 3′ direction, while the other has the opposite 3′ to 5′ polarity. DNA Pol III synthesizes DNA in only the 5′ to 3′ direction, therefore the direction of synthesis on both strands is opposite to each other.

One strand is synthesized towards the replication fork is called the leading strand having continuous DNA. The other strand that is synthesized in the opposite direction to the replication fork is called the lagging strand. The synthesis of this strand requires many primers and, therefore, has DNA fragments with intervening primers. These pieces are called Okazaki fragments because the evidence supporting the discontinuous DNA synthesis was first provided by Reiji and Tuneko Okazaki. The length of the fragments is 1000 to 2000 nucleotides.  

 f.      Removal of primer

The lagging strand has discontinuous DNA due to the presence of primers. These primers are removed by the DNA polymerase-I enzymes. This enzyme has the 5’ exonuclease and 3’ polymerase activity removing nucleotides one by one from the 5’ end of the primer and adding nucleotides to the 3’ end of the Okazaki fragment.

 g.     Nick sealing

After the replacement of primers, The DNA ligase enzyme joins the two DNA fragments by catalyzing the formation of the phosphodiester bond that seals the nick between the discontinuously synthesized strands.

Proofreading and Error Correction

Although the action of DNA polymerases is accurate, sometimes synthesis is not perfect and a noncomplementary nucleotide is occasionally inserted.  To remove these nucleotides the DNA polymerases possess 3′ to 5′ exonuclease activity. These enzymes detect and excise a mismatched nucleotide (in the 3′ to 5′ direction). Once the mismatched nucleotide is removed, 5′ to 3′ synthesis can again proceed.