DNA Replication
Assessment Statement
7.2.1 State that DNA replication occurs in a 5’ – 3’ direction
7.2.2 Explain the process of DNA replication in prokaryotes, including the role of
enzymes (helicase, DNA polymerase, RNA primase, and DNA ligase),
Okazaki fragments and deoxynucleoside triphosphates
7.2.3 State that DNA replication is initiated at many points in eukaryotic
chromosomes
Before mitosis occurs, the DNA is replicated during Interphase – the portion of time the
cell “lives”.
When Watson and Crick proposed their model for the structure of DNA, they realized
that the A-T and G-C base pairing provided a way for DNA to be copied, as a single
strand could serve as a template to form a copy.
The actual process of DNA replication was first shown in 1957 by Meselson and Stahl
from the California Institute of Technology. What follows is their experiment:
It is possible to grow bacteria in a medium (agar) that contains a heavy isotope of
nitrogen, 15N. If the agar does not contain any other nitrogen, the bacteria must
incorporate this nitrogen into its nitrogen bases, and therefore, it’s DNA. DNA with the
heavy form of nitrogen can be detected as it responds differently when centrifuged.
- Meselson and Stahl grew cultures of E. coli bacteria in the 15N medium.
- The resulted in all bacteria having heavy nitrogen incorporated into their DNA
- They then moved the cultures to a new medium that contained only 14N.
- The first generation on the new plate all had half 15N and half 14N DNA – this was
called hybrid DNA
- The second generation showed half the cells with hybrid DNA, and half with light
DNA (only 14N)
- The third generation showed ¼ hybrid DNA, with ¾ light DNA
The above results of the experiment support one explanation: The replication of DNA is
a semi-conservative process. If DNA replication had followed a conservative process,
(keeping the original strand intact and forming a complete new one), what would the
results have been in the first generation?
This must mean that for DNA replication to occur, the DNA must unwind and then
“unzip”, or have the hydrogen bonds between nitrogen bases undone, before replication
occurs. The enzyme helicase is responsible for this.
Replication in General
The replication of DNA begins at special sites called origins of replication. Bacterial, or
prokaryotic DNA is circular and has a single origin. Eukaryotic DNA is linear and has
thousands of origins. This accelerates the time taken to replicate large eukaryotic
chromosomes.
The general process of replication is as follows:
- Replication begins at the origin, which appears as a bubble, because the strand
separates, unwinds, and is unzipped by helicase
- At each end of the bubble there is a replication fork. This is where the two
parental DNA strands open to provide templates for the semi-conservative
replication. Free nucleotides floating around the nucleus form complimentary
bonds with the nucleotides of the DNA strands (both strands), then join to form a
new strand of DNA – this is accomplished by DNA polymerase
- The bubbles enlarge in both directions and eventually fuse. The result is 2
identical double helices, with one old and one new strand
Replication in Detail
As we know from the previous topic, DNA strands are either 3’ – 5’ or 5’ – 3’ depending
which strand in the helix is referenced. This is very important when it comes to
replication, because with the 2 strands running in opposite directions, a slightly different
technique must be used for replication to take place.
DNA polymerase creates the 3’ – 5’ linkages between the nucleotides thus creating a new
DNA strand, complimentary to the original one and identical to the one that was
unzipped. DNA polymerase creates the covalent bonds between the nucleotide of the
growing strand.
Since DNA is in a 3’ – 5’ linkage, the DNA replication occurs in a 5’ – 3’ direction.
But what does this mean for a strand being formed in the opposite direction? Well,
because DNA polymerase does not work in this direction, it lags behind the other when
forming, and is therefore called the lagging strand. The lagging strand forms Okazaki
fragments of between 100 and 200 nucleotides, which are later bound together by ligase.
*Remember – DNA replication takes place at several places along the DNA double helix
at the same time! To replicate DNA in a semi conservative process, the following steps
need to be taken.
Process
1. The DNA is unwound by the enzyme helicase
2. Helicase then also breaks the hydrogen bonds between the two strands, separating
the strands. One of the old strands will be 3’ – 5’ and the other will be 5’ – 3’
3. There are deoxyribonucleosides triphosphates (dNTP) present in the nucleus.
These are an organic base, a deoxyribose sugar and three phosphate groups, and
are sometimes referred to as dATP, dCTP, dGTP, and dTTP, the precursors to the
nucleotides that will make up the new strands of DNA
4. Before the new strands begin to form, it is necessary to start with an RNA primer.
The RNA primer is a few (5-10) RNA nucleotides, which bind to the old DNA
strand (by hydrogen bonding). The enzyme RNA primase will bind the RNA
nucleotides together.
5. Now that the RNA primer is in place, the dATP’s etc will form hydrogen bonds
between their organic bases and the complimentary base on the exposed strand of
the old DNA. For example, if the template strand has T, then dATP will bond to
form the A – T bond.
6. In the new strand that is forming in the 5’ – 3’ direction, (this is also known as the
leading strand) DNA polymerase III will bind the new nucleotides to the
growing strand by covalent bonds formed via condensation reactions. DNA
polymerase III only works in the 5’ – 3’ direction!
7. As it attaches to the growing DNA strand, the 2nd and 3rd phosphate groups are
removed from the DATP’s etc, changing it into a nucleotide
8. DNA polymerase I will then remove the RNA primer and replace the RNA
nucleotides with DNA nucleotides
9. The other strand is in 3’ – 5’, and the polymerase works in a 5’-3’ direction. This
means the DNA cannot be made in one piece, but several pieces. As a result, the
process “lags” behind the process. Okazaki fragments must be made. On the
lagging strand, the Okazaki fragments form in much the same way
10. RNA primer is formed from RNA and RNA primase
11. dATP’s etc form hydrogen bonds to complimentary bases of old DNA
12. DNA polymerase III bonds the DNA nucleotides to form the Okazaki fragments
13. DNA polymerase I will then remove the RNA primer and replace the RNA
nucleotides with DNA nucleotides
14. DNA ligase then forms the bonds attaching the DNA fragments
Remember that the DNA of prokaryotes is circular, and occurs from one point, as in the
handout. Eukaryotic DNA Replication is initiated at several points along the
chromosome, due to the size.
Even though the process seems quite complicated, it occurs at a speed of 4000
nucleotides per second. The process is also quite accurate. Mutations are few and those
errors can be corrected by repair enzymes in the cell.