LECTURE 8: DNA REPLICATION

Introduction
▪ Prokaryotes e.g., bacteria, and eukaryotic cells e.g., mammalian cells reproduce by binary
fission in a process where one cell divides to give two identical daughter cells.
▪ Most of the genetic information in bacteria is found on a single circular chromosome which
consists of DNA and proteins. In eukaryotic cells, the genetic material is organized in several
chromosomes depending on the species e.g., in a human somatic cell’s DNA is packaged into
23 pairs of chromosomes. During cell division, this information must be distributed equally
to each of the daughter cells.
▪ This is only possible because DNA is a self-replicating molecule and can make an exact copy
of itself before cell division occurs. One copy will go to each daughter cell.
▪ The structure of DNA plays an important role in the replication process
▪ Complementation: Adenine pairs with Thymine, while Cytosine pairs with Guanine,
o The two strands of DNA are held together by hydrogen bonds that exist between
complementary bases.
o Anti-parallel: One strand runs in the 5' to 3' direction, the other in the 3' to 5'
direction.

DNA Replication
▪ Replication of DNA occurs during the process of normal cell division cycles. The daughter
cells must have the same genetic information as the parental cell. So DNA replication must
possess a very high degree of fidelity.
▪ The entire process of DNA replication is complex and involves multiple enzymatic
activities.
▪ DNA exists in the nucleus as a condensed, compact structure -CHROMOSOMES.

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▪ To prepare DNA for replication, a series of proteins and enzymes are required. These aid in
the unwinding and separation of the double-stranded DNA molecule as well as
CATALYTIC activity.
▪ These proteins are required because DNA must be single-stranded before replication can
proceed.
Proteins of DNA Replication
i) DNA Helicases
▪ These proteins bind to the double-stranded DNA and stimulate the separation of the
two strands.
(ii) DNA single-stranded binding proteins
▪ These proteins bind to the DNA as a tetramer and stabilize the single-stranded
structure that is generated by the action of the helicases.
▪ Replication is 100 times faster when these proteins are attached to the single-
stranded DNA.
iii) DNA Polymerase
▪ DNA polymerase catalyzes the synthesis of a new strand of DNA.
▪ They require an RNA primer with a free 3'-OH group.
▪ They use deoxyribonucleoside-5-triphosphate precursors.
DNA polymerases of prokaryotes
DNA replication was originally characterized in the bacterium, Escherichia coli which
contains 3 distinct enzymes.
a. DNA polymerase I was the first enzyme to be discovered and three activities are
associated with it;
▪ DNA polymerase I primary role is to ensure the fidelity of replication through the
repair of damaged and mismatched DNA.
1. 5' ----> 3' elongation (polymerase activity)
2. 3' ----> 5' exonuclease (proof-reading activity)
3. 5' ----> 3' exonuclease (repair activity)
NB: The second two activities of DNA Pol I are important for replication.
b. DNA Topoisomerases
▪ The progression of the replication requires that the DNA is continuously unwound.
▪ Due to the fact that eukaryotic chromosomal DNA is attached to a protein the
replication fork introduces severe stress into the duplex ahead of the fork.
▪ This stress is relieved by DNA topoisomerases.
▪ The nicks are then resealed by the topoisomerases.
c. DNA Gyrase
▪ This enzyme catalyzes the formation of negative supercoils that is thought to aid with
the unwinding process.
▪ The enzyme is a target of several antibiotics such as ciprofloxacin, nalidixic acid,
and novobiocin used against UTI and other infections
d. DNA Ligases
▪ Nicks occur in the developing molecule because the RNA primer is removed
▪ Synthesis proceeds in a discontinuous manner on the lagging strand and these pieces
are joined by the ligase enzyme.
▪ The gaps that exist are repaired by DNA ligases
e. Primase

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 ▪ Their function is to synthesize RNA primers that are required at the initiation sites
of replication.
▪ After replication the RNA primers are removed and the repair DNA polymerases
simultaneously replace the ribonucleotides with deoxyribonucleotides.
▪ The final replication product does not have any nicks because DNA ligase forms a
covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.
Mechanism of DNA Replication in prokaryotes
▪ DNA replication begins with a partial unwinding of the double helix at an area known as
the origin of replication or the replication fork.
Illustration of replication fork

▪ This unwinding is accomplished by the enzyme known as DNA helicase.

▪ This unwound section appears under electron microscopes as a "bubble" and is thus known
as a replication bubble.
▪ As the two DNA strands separate and the bases are exposed, the enzyme DNA polymerase
moves into position at the point where synthesis will begin.
▪ The starting point for DNA polymerase is a short segment of RNA known as an RNA
primer.
▪ The very term "primer" is indicative of its role which is to "prime" or start DNA synthesis
at certain points.
▪ The primer is "laid down" complementary to the DNA template by an enzyme known as
Primase.

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▪ The DNA polymerase (once it has reached its starting point as indicated by the primer) then
adds nucleotides one by one in an exactly complementary manner, A to T and G to C.
Addition of bases
▪ DNA polymerase is described as being "template-dependent" in that it will "read" the
sequence of bases on the template strand and then "synthesize" the complementary strand.
▪ The template strand is ALWAYS read in the 3'---> 5' direction (that is, starting from the 3'
end of the template towards the 5' end of the template).
▪ As a result, the new DNA strands can grow only in the 5' to 3' direction, and strand growth
must begin at the 3' end of the template
DNA Replication is Semi-conservative
▪ When the replication process is complete, two DNA molecules - identical to each other and
identical to the original - have been produced.
▪ Each strand of the original molecule has remained intact as it served as the template for the
synthesis of a complementary strand.
▪ This mode of replication is described as semi-conservative: one-half of each new molecule
of DNA is old; one-half new

RNA dependent synthesis of RNA and DNA

Reverse transcriptase
▪ Certain animal viruses contain a unique enzyme RNA-directed DNA polymerase
called reverse transcriptase
▪ The single-stranded RNA strand is used to synthesize a complementary DNA
strand. The DNA is incorporated into the host genome
▪ These viruses are known as retroviruses
Anti-retrovirals nucleotide analogs
▪ Nucleotide analogues also have been targeted for use as antiviral agents.
▪ Several analogs are used to interfere with the replication of HIV:-AZT
(azidothymidine) and ddI (dideoxyinosine)

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 Eukaryotic DNA replication process
▪ The large size of eukaryotic chromosomes makes it necessary for multiple origins of
replication to exist in order to complete replication in a reasonable period of time.
▪ The precise nature of the origins of replication in higher eukaryotic organisms is
unclear.
▪ However, it is clear that at a replication origin, the strands of DNA must dissociate
and unwind in order to allow access to DNA polymerase.
Speed of replication: Eukaryotes
▪ The average human chromosome contains 3.0 x 109 nucleotide pairs which are copied
at about 50 base pairs per second.
▪ The process would take a month but there are many places on the eukaryotic
chromosome where replication can begin (many origins)
▪ As replication nears completion, "bubbles" of newly replicated DNA meet and fuse
Illustration of Replication in eukaryotes

Post-Replication Modification of DNA

▪ One of the major post-replicative reactions that modify the DNA is methylation.
▪ The sites of natural methylation of eukaryotic DNA are always on cytosine residues that are
present in C-G dinucleotides. However, not all C-G dinucleotides are methylated at the C
residue.
▪ This methylation serves a structural as well as a regulatory function. The cytidine is
methylated at the 5 position of the pyrimidine ring generating 5-methylcytidine.
▪ Methylation of DNA in prokaryotic cells also occurs.
o The function of this methylation is to prevent degradation of host DNA by
endonucleases.
o Bacteria have enzymes called restriction endonucleases that degrade foreign DNA
(methylation protects bacteria DNA from degradation by the restriction
endonucleases)
o Restriction endonucleases recognize specific nucleotide sequences of DNA
o The role of this system in prokaryotic cells (called the restriction-modification
system) is to degrade invading viral DNAs.
o Since the viral DNAs are not modified by methylation they are degraded by the host
restriction enzymes.
o The methylated host genome is resistant to the action of these enzymes.

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POLYMERASE CHAIN REACTION (PCR)

▪ The polymerase chain reaction (PCR) makes many copies of a gene or other DNA segment.
This might be done to make large quantities of a gene for genetic testing.
▪

▪ Components of PCR, such as template DNA, primers, nucleotides, and polymerase, are
assembled in a tube.
▪ The tube is inserted into a machine (thermocycler) that controls and alters the temperature of
the tube during PCR.
▪ PCR involves three steps: denaturing, annealing, and extension.
1. In the first step - denaturation, the temperature is increased to 94 - 96ºC to denature the
template. Denaturing involves heating DNA to break the bonds holding together the two
DNA strands. This yields two single strands of DNA.
2. In the second step, annealing involves cooling the single strands of DNA and mixing them
with short DNA segments called primers. Primers have base sequences that are
complementary to segments of the single DNA strands. As a result, bonds form between the
DNA strands and primers.
3. In the third step - elongation, the temperature is increased to about 72ºC, so Taq polymerase
can extend the strand from primer by adding nucleotides to create new DNA fragments. The
enzyme Taq polymerase is used in PCR because it can withstand high temperatures.
▪ These three steps are repeated multiple times to create exponential copies of the targeted
DNA fragment.