2 Replication of DNA
(a) Replication of DNA by DNA polymerase and primer. Directionality of replication on both template strands. DNA polymerase adds complementary nucleotides to the deoxyribose (3′) end of a DNA strand. Fragments of DNA are joined together by ligase.
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abridged from: Major Molecular Events of DNA Replication By: Leslie A. Pray, Ph.D. © 2008 Nature Education
A typical bacterial cell has anywhere from about 1 million to 4 million base pairs of DNA which require to be replicated prior to cell division. The events of DNA replication are divided into three major stages: unwinding, primer synthesis, and elongation.
Unwinding
DNA replication in prokaryotes starts at the origin of replication. At this site, helped by several enzymes (including helicase) and other proteins, the DNA double helix unwinds into two single-stranded DNA molecules. Because of the characteristic y-shape of the replicating DNA, it is often referred to as a “replication fork.” DNA replication can only occur in the 5′ to 3′ direction, and so whilst replication proceeds along the two single strands at the same time, DNA replication is in opposite directions (i.e., left to right on one strand, and right to left on the other).
Primer Synthesis
Primer synthesis marks the beginning of the actual synthesis of the new DNA molecule. Primers are short stretches of RNA nucleotides (about 10 to 12 bases in length) synthesized by an RNA polymerase enzyme called primase. Primers are required because DNA polymerases, the enzymes responsible for the actual addition of nucleotides to the new DNA strand, can only add deoxyribonucleotides to the 3′-OH group of an existing chain and cannot start chains. Primase, on the other hand, can start chains. Later, after elongation is complete, the primer is removed and replaced with DNA nucleotides.
Elongation
Elongation–the addition of nucleotides to the new DNA strand–begins after the primer has been added. Synthesis of the growing strand involves adding nucleotides, one by one, in the exact order specified by the original (template) strand. According to Chargaff’s rules, adenine always pairs with thymine and cytosine always pairs with guanine.
DNA is always synthesized in the 5′-to-3′ direction , meaning that nucleotides are added only to the 3′ end of the growing strand. This is because nucleotide triphosphates are used in the synthesis of the DNA strand and the position of the tri-phosphate group on the 5′ carbon means that DNA can only be synthesised by adding to the 3′ end (an explanation of why, useful for chemistry students, can be found here). As shown in the figure above, the 5′-phosphate group of the new nucleotide binds to the 3′-OH group of the last nucleotide of the growing strand. The energy required to drive the reaction (DNA replication) is provided by the break of the two phosphates from the nucleotide triphosphate (similar to breaking ATP!)
However, as DNA is an anti-parallel molecule, the DNA code in each of the newly synthesised strands, whilst complementary to the template strand, runs in the opposite direction and so, because DNA is anti-parallel, one of the new strands needs to be synthesised in the opposite direction. The cell overcomes this difficulty by creating a leading strand, synthesised in the 5′ to 3′ direction and a lagging strand. The lagging strand is generated from a series of smaller fragments (see Figure), known as Okazaki fragments, also synthesised in the 5′ to 3′ direction . These fragments are subsequently joined together by the enzyme, DNA ligase, to form a long continuous DNA strand.
The following video will help you understand this area. (skip to 9 minutes in for DNA replication)
The Challenges of Eukaryotic Replication
Bacterial and eukaryotic cells share many of the same basic features of replication; for instance, unwinding the DNA is accomplished by an enzyme named DNA helicase and manufacturing new DNA strands by enzymes called polymerases. Both types of organisms also follow a pattern called semi-conservative replication. Initiation requires an RNA primer and elongation is always in the 5′-to-3′ direction, continuous along the leading strand and discontinuous along the lagging strand by the production of small DNA fragments called Okazaki fragments that are eventually joined together.
Differences between Prokaryotic and Eukaryotic DNA Replication
The differences between prokaryotic and eukaryotic DNA replication are largely related to the size of the genomes of the two types of cell. The average eukaryotic cell has 25 times more DNA than a prokaryotic cell. In prokaryotic cells, there is only one point of origin. DNA replication moves from this origin in both directions (left and right).
Eukaryotic cells on the other hand, have multiple points of origin replicatiing in one direction only.
Replication also happens at a much faster rate in prokaryotic cells, than in eukaryotes.
In E. coli, the entire genome is replicated in just 40 minutes, at a pace of approximately 1,000 nucleotides per second. In eukaryotes, the pace is much slower: about 40 nucleotides per second, but their multiple points of replication mean they can accomplish DNA replication faster than these numbers would suggest.
Bacterial chromosomes are circular, eukaryotic chromosomes are linear. During circular DNA replication, the primer is easily replaced, leaving no gap in the newly synthesized DNA. In contrast, in linear DNA replication, a small gap at the very end of the chromosome cannot be filled because of the lack of a 3′-OH group for nucleotides to bind on to. (As mentioned, DNA synthesis can proceed only in the 5′-to-3′ direction.) If there were no way to fill this gap, the DNA molecule would get shorter and shorter with every generation. However, the ends of linear chromosomes—the // telomeres—have several properties that prevent this.
During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened[1] (this is because the synthesis of Okazaki fragments requires RNA primers attaching ahead on the lagging strand). The telomeres are disposable buffers at the ends of chromosomes which are truncated during cell division; their presence protects the genes before them on the chromosome from being truncated instead.
Over time, due to each cell division, the telomere ends become shorter.[2] They are replenished by an enzyme, telomerase reverse transcriptase.
The details of DNA replication were worked out by Meselson and Stahl, using different nitrogem isotopes(14N, 15N). They followed the inheritance of heavy (15N) DNA into the next generation, and found that the two daughter cells each inherited one heavy strand intact (from the previous generation) and a newly synthesised, light strand (14N). Their results demonstrated that DNA replication was semi-conservative. Details of their experiments can be found by clicking the image.
