Learning Unit 3

DNA replication

By MS AE KANEME
 DNA Replication Introduction
• Genetic material must be copied for transmission of genetic material
to occur.
• Process known as DNA replication.
• Original strands used as templates for the synthesis of new strands.
• Bacterial DNA replication.
• Occurs very quickly and accurately.
• Cellular proteins play a vital role
• Need to understand the mechanism of DNA replication and the
proteins involved.
 Structural overview of DNA replication
• Double helix has two DNA strands.
• Nucelotides are the building blocks of the strands.
• The bases are A, T, C or G.
• Double strand held together by base stacking by hydrogen bonds that
hold the complementary nucleotides together.
• A – T C-G
• Antiparallel alignment
• Direction important when considering the enzymes that synthesis
new DNA strands.
 Templates for synthesis
• DNA replication relies on the complementarity of DNA strands according to the AT/GC
rule.
• The two complementary strands come apart and serve as template strands or parental
strands.
• The double helix starts to separate and the individual nucleotides have access to the
template strands.
• Hydrogen bonding then occurs between the nucleotides which are complementary.
• Covalent bond forms between the phosphate of one nucleotide and the sugar of the
previous nucleotide.
• The two newly made strands are called the daughter strands.
• Bases are identical in both daughter strands.
• Retain the same information.
Mechanism for DNA replication
 Models of DNA replication

There are 3 different Models for DNA

replication. These are:
1. Conservative model – both strands of
parental DNA remain together following DNA
replication
2. Semi-conservative model – the newly made
double-stranded DNA contains one parental
strand and one daughter strand
3. Dispersive model – segments of parental
DNA and newly made DNA are interspersed in
both strands following replication.
 Evidence that DNA replication is
semiconservative
• Matthew Meselson and
Franklin Stahl
• Distinguished between
newly made daughter
strands and the original
parent strand.
• In 1958 Meselson and Stahl devised a method to experimentally distinguish newly made daughter strands of
DNA from the original parents. Their approach required the use of heavy isotope labelling. Nitrogen in the
bases of DNA can occur as a light 14N or a heavy 15N form. Prior to the experiment, they grew E. coli in a
heavy 15N form for generations. This produced cells whose nitrogen in their DNA was in the form 15N.
Procedure they followed:
• 1. They grew the E. coli whose DNA consisted of the heavy 15N form in media with the light 14N form.
• 2. After a series of time e.g 30min, 1Hr, e.t.c in the media containing only the light 14N formed.
• 3. After the given time point DNA was extracted from the microorganisms and the lysate was loaded onto a
Cesium Chloride (CsCl) gradient.
• 4. The DNA in the CsCl gradient was centrifuged until the DNA molecules reached their densities
• 5. DNA with a gradient was then observed under UV light.

• For them to prove that DNA replication was semi-conservative they needed to see 3 band these were
• i) heavy DNA at the bottom (from the cells that had not replicated and only had the heavy 15N form )
• ii) half heavy DNA (in the middle from the cells that had replicated for more than 2 generations and had
both the heavy 15N and the light 14N forms)
• iii) light DNA (on top from the cells that had replicated for more than 4 generations and had majority of the
light 14N form).
 Learning Unit 3
DNA replication in
Bacteria
 Single origin of Replication
• Site on bacterial chromosome where DNA synthesis begins is called
the origin of replication.
• Bacterial chromosomes have only a single origin of replication.
• Bidirectional – synthesis is initiated with in the origin
• two replication forks which move in opposite directions outwards
from the origin are called Bidirectional replication.
• Replication fork is the site where the parental DNA strands have
separated, and new daughter strands are being made.
STEPS INVOLVED IN BACTERIAL
REPLICATION
1. Initiation
2. Elongation
3. Termination
 Initiation of Replication
• OriC – origin of chromosome replication.
• Three DNA sequences within oriC – AT rich region, DnaA box and GATC
methylation site.
• Initiation of replication occurs within OriC by the binding of DnaA proteins to
sequences within the origin known as DnaA box sequences.
• DnaA box sequences serve as recognition sites for the binding of DnaA proteins.
• They bind to five DnaA boxes in oriC to initiate replication.
• They form complex.
• Results in the separation of the AT-rich region – only have two Hydrogen bonds
with A and T base pairs and therefore easily separated.
• Following separation of the strands, the DnaA proteins with the help of DnaC
proteins recruit DNA Helicase proteins to the site.
• DnaB also called helicase.
• When a DNA helicase finds a double stranded region, it breaks the H bonds
between the two strands.
• This generates two separate strands.
• Two helicase required – separate the strands at the oriC region and beyond.
• Proteins use the energy from ATP hydrolysis to catalyze the separation of the
double stranded parental DNA.
• Helicase binds to the single stranded DNA and travels along the DNA in a 5’ to 3’
direction.
• This keeps the replication fork moving.
• DNA helicase promoted the movement of the two replication forks .
• This initiates the replication in both directions and therefore termed bidirectional
replication.
 Elongation:
Functions of Proteins involved in DNA replication
at the replication fork
• The strands must separate for them to act as templates.
• DNA helicase breaks the H bonds between the DNA strands to generates
positive supercoiling.
• DNA gyrase or Topoisomerase – alleviates positive supercoiling
• Keep the strands separated to prevent the DNA strands from coming
together.
• Single stranded binding proteins – keep strands separated. Prevents
reforming of double helix.
• Hence the parental strands remain exposed this ensure the nucleotides for
the new strand to bind.
• RNA primers – short strands of RNA.
• Synthesized by the linkage of ribonucleotides by an enzyme known as
primase.
• This enzymes synthesizes short strands of RNA – 10 -12 nucleotides in
length.
• These short strands (Leading and Lagging strand) start or prime the
process of DNA replication.
• Leading strand – single primer made at the origin of replication
• Lagging strand – multiple primers are made
• DNA polymerase – synthesis of the DNA of the leading and lagging
strand.
• Catalyzes the formation of the covalent bonds between the adjacent
nucleotides and thereby makes a new daughter strand.
• In bacterial, five different proteins -Polymerase I, II, III, IV and V-
function as DNA polymerases.
• Polymerase I and III involved in normal DNA replication
• Polymerase II, IV and V involved in DNA repair and replication of
damaged DNA.
• DNA polymerase III responsible for most of the DNA replication.
• It is a large enzyme consisting of 10 different subunits that plays vital
roles in replication process.
• DNA Polymerase I is composed of a single subunit and plays a role in
removal of RNA primers and fill in the vacant regions with DNA.
• The catalytic subunits of all DNA polymerases has a structure that
resembles the human hand in shape (Fig 11.8 page 278).
• The template DNA is thread through the palm of the hand, the thumb
and fingers are wrapped around the DNA.
• The incoming dNTPs enter the catalytic site, bind to the template strand
and then covalently attach to the 3’ end of the growing strand.
• DNA polymerase also can remove mismatched bases.
Proteins involved in DNA replication
Two Unusual features of DNA polymerase function:
1. DNA polymerase can elongate a strand only from RNA primer or existing
DNA strand.
2. DNA polymerase can only attach nucleotides in the 5’ to 3’ direction not
in the other direction.

• Why is RNA primer required?

• DNA polymerase cannot add the first individual nucleotides and therefore cannot
start DNA synthesis.
• This enzyme can only elongate a preexisting strand starting with an RNA primer
preexisting DNA strand.
• Because of these two features the synthesis of the leading and lagging
strands show difference in their synthesis.
 Synthesis of the Leading Strand

• In the leading strand one primer is made at the origin and then the
DNA polymerase II can attach nucleotides in a 5’ to 3’ direction as it
slides toward the opening of the replication fork.

• We therefore say that the replication is continuous.

 Synthesis of the Lagging Strand
• Lagging strand – the synthesis of DNA elongates in a 5’ to 3’ direction
but it does in the direction away from the replication fork
• The RNA primers must repeatedly initiate the synthesis of the short
segments of DNA. Therefore, the synthesis is said to be discontinuous.
• The length of the fragments are 1000 – 2000 nt.
• Each fragments contains a short RNA primer at the 5’ end which is
made by the enzyme primase.
• These short DNA fragments that are made are called Okazaki
fragments.
• To complete the synthesis of the Okazaki fragments within the lagging strand, three
events must first occur:
• Removal of the primers
• Synthesis of the DNA in the areas where the primers have been removed.
• Covalent attachment of adjacent fragments of DNA.
• DNA polymerase I removes the RNA primers.
• Has a 5’ to 3’ exonuclease activity – digests away the RNA primers leaving a vacant
region.
• DNA polymerase I would remove the RNA primer from the first Okazaki fragment and
then synthesize DNA in the vacant region by attaching nucleotides to the 3’ end of the
second Okazaki fragment.
• DNA ligase – catalyzes the formation of a covalent bond between adjacent fragments to
complete the replication process in the lagging strand.
• Figure 11.10 page 279 – summary of the process of DNA synthesis at the replication fork.
 Diff between leading and lagging Similarities bw leading and lagging
strands strands

Leading strand Lagging strand Leading strand Lagging strand

Replicates continuously Replicates discontinuously DNA polymerase adds DNA polymerase adds
nucleotides nucleotides
Adds nucleotides to the 3' Adds nucleotides to the 5' Helicase unwinds DNA helix Helicase unwinds DNA helix
end of the growing strand end of the growing strand
single primer made at the multiple primer made Replicated strands of DNA Replicated strands of DNA
origin of replication
Okazaki fragments are not Okazaki fragments are
formed and DNA ligases are formed which are joined by
not required DNA ligase
Helicase and Topoisomerase work together to expose single-strand DNA templates. Primase adds RNA
nucleotides, reading the old DNA single-strand template 3′ to 5′. Image by Walter Suza.
DNA Polymerase III can add to the 3′ end of the RNA primer and start to read the template 3′ to 5′. Primase can
prime the other template strand at this replication fork. Image by Walter Suza.
Both leading and lagging strands are being made at this replication fork. DNA polymerase I removes
RNA primer nucleotides and replaces them with DNA. Image by Walter Suza.
Lagging strand synthesis must be primed multiple times with primase enzyme creating discontinuous
replication. Leading strand just needs to be primed one time. Image by Walter Suza.
Ligase must form a sugar-phosphate bond between the last nucleotide placed by DNA pol I and the first
nucleotide placed by DNA pol III. Image by Walter Suza.
Short fragments made by discontinuous replication were discovered by the Okazaki team and are called
the Okazaki fragments. Image by Walter Suza
 DNA Polymerase III
• Catalyzes the synthesis of daughter
strand very quickly – 750 nucleotide
per second.
• Processive enzyme.
 Termination of Replication
• Termination sequences – ter sequences.
• A protein known as termination utilization substances (Tus) binds to
the ter sequences and stops the movement of the replication fork.
• Prevents the advancement of the termination fork moving left to
right.
• Following termination DNA ligase covalently links the two daughter
strands creating two circular double-stranded molecules.
• Topoisomerase II introduces a temporary break into the DNA strands
and then rejoins them. This allows the catenanes to be separated
into individual circular molecules.
Processive Enzymes and Proof Reading

• Processive enzymes are very important in DNA replication.

• processive enzyme, such as DNA or RNA polymerase, glides along the DNA and does not dissociate from the
template strand as it catalyzes the covalent attachment of nucleotides
• Proofreading function – the ability of DNA polymerase to remove mismatched bases from a newly made
strand. The DNA polymerase pauses when there is a mismatch so that the incorrect nucleotide is at the 3’
exonuclease site. It then goes back (3’to 5’ direction) and removes this mismatched nucleotide by digesting
it. The polymerase then resumes with addition of nucleotides in the 5’ to 3’ direction
• Co-ordination of DNA replication and Cell Division
• DNA replication should take place only when a cell is ready to divide. If it occurs too frequently there will be
too many copies of the chromosomal DNA (chromosome 21). If it doesn’t occur often enough then a
daughter cell could end up without a chromosome. Bacterial cells regulate DNA replication by controlling the
initiation of replication at oriC
 Home work
1. Briefly explain the fidelity of DNA replication
and how this is ensured based on the proofreading
mechanism.
• One way that DNA polymerase decreases
the error rate during DNA synthesis is by enzymatic
removal of mismatched nucleotides
• The ability to remove mismatched bases through this
mechanism is called the proofreading function
2.How is bacterial DNA replication coordinated with
Cell division?

• Bacterial cells regulate the DNA replication process by controlling the

initiation of replication at OriC
• After the initiation of DNA replication, the DNaA protein hydrolyzes its
ATP , and switches to an ADP-bound form which has a lower affinity to
DNaA boxes thus preventing premature initiation
• Insufficient amount of DNaA protein
• Methylation of GATC sites at OriC
Replication concept Check
•1. What is the purpose, outcome of DNA replication AND what stage
does it happen in the cell cycle?
•To create an identical, duplicate copy of original DNA, “S”
•2. What enzyme is responsible for “unzipping” the DNA double
helix?
•DNA Helicase
•3. What enzyme is responsible for adding nucleotides to the
“unzipped” DNA?
•DNA polymerase
•4. What is the enzyme responsible for bonding the fragments on the
lagging strand?
•Ligase
•5. What is the enzyme responsible for “proofreading” the newly
made DNA, checking for mismatched base-pairs?
•DNA polymerase
Homework