RECOMBINANT DNA202

DNA REPLICATION

Dr Danisile Tembe, SLS Room 05-105, Email:

TembeD@ukzn.ac.za
This Chapter Focuses On The Process Of DNA
Replication
 DNA REPLICATION
➢The process in which two identical daughter DNA are produced from parental DNA
molecule

• DNA replication happens so that daughter cells get the exact copies as that of the
original cell

• Without DNA replication there could be no transfer of information across

generations

• Replication should occur only when the cells has sufficient resources to divide and
form two new cells

• To avoid cases where one cell will have incomplete information from the
parental strand while the other have competed information
 DNA REPLICATION

➢Daughter DNAs are produced using parental DNA as a template

Parental DNA Replication Daughter DNA

 3 PROPOSED MODELS OF REPLICATION

➢ Semiconservative

➢Conservatives

➢ Dispersive
3 PROPOSED MODELS OF REPLICATION
 DNA REPLICATION IS SEMICONSERVATIVE

➢Watson and Crick proposed that the model of DNA

replication is semiconservative

• Base pairing allows each strand to serve as a

template for a new strand

➢Semi-conservative: because one strand of the

parental DNA is conserved in each daughter
molecule
 DNA REPLICATION IN PROKARYOTES

➢Several enzymes and proteins participate in DNA replication

• DNA polymerases:

• E. coli contains three different DNA polymerases

• DNA polymerase I - repairs DNA and participates in the synthesis of one of

the strands of DNA during replication

• DNA polymerase II - plays a role in DNA repair

• DNA polymerase III - is the major DNA replication enzyme

• It is responsible for chain elongation during DNA replication

DNA REPLICATION IN PROKARYOTES
 STEP 1: UNWINDING

➢ Bacteria- replication of chromosomal DNA begins at origin of replication – short

stretches of DNA with specific sequence of nucleotide

• E. coli has a circular chromosome and has a single origin of replication

 STEP 1: UNWINDING

➢ Proteins that initiates replication recognize origin of replication, attach to the DNA &
separate two double strands,

➢ Forming a replication bubble, at each end of replication bubble there is a replication

fork
 STEP 2: DNA SYNTHESIS

➢ Replication proceeds in both directions until the forks meet resulting to two daughter
cells
DNA REPLICATION IN
EUKARYOTES
 UNWINDING AND DNA SYNTHESIS

➢ Eukaryotes chromosome have multiple origins of replication

➢ Proteins that initiates replication recognize origins of replication
➢ Multiple replication bubbles form at multiple sites & fuse, resulting into two linear daughter
cells
 SYNTHESIS OF TWO DAUGHTER CELLS

➢ Several proteins involved in the unwinding of

DNA strands

• Helicase
• Single strand binding proteins
• Topoisomerase
 SYNTHESIS OF TWO DAUGHTER CELLS

➢ Several proteins involved in the unwinding of

DNA strands

• Helicase
• Single strand binding proteins
• Topoisomerase
• Primase
REPLICATION: 1ST STEP
➢ Unwind DNA:

➢ Helicase enzyme : unwinds DNA helix

• stabilized by single-stranded binding proteins

• prevents DNA molecule from binding

➢ DNA gyrase

• Enzyme that prevents tangling upstream from the replication fork

REPLICATION: 2ND STEP

➢ RNA Primase

➢ Adds small section of RNA (RNA primer) to the 3’ end of template DNA

➢ Why must this be done?

➢ DNA polymerase 3 can only add nucleotides to existing strands of DNA

NB: DNA SYTHESIS REQUIRES RNA PRIMER

REPLICATION: THIRD STEP

➢ Build daughter DNA strand

➢ add new complementary bases

➢ With the help of the enzyme DNA polymerase III

REPLICATION: 4TH STEP

➢ Replacement of RNA primer

➢ With DNA

➢ Done by DNA Polymerase l

➢ Ligase to seal the gaps

 SYNTHESIS OF TWO DAUGHTER CELLS

➢ The unwound strands are available as template strands for the synthesis of
new complementary strands

➢ However, the enzymes that synthesize DNA, cannot initiate the synthesis of
polynucleotide

• They can only add nucleotides to the end of a primer, paired with the
template strand
 SYNTHESIS OF TWO DAUGHTER CELLS AT A SINGLE
REPLICATION FORK

➢ An initial chain that is used as a pre-existing chain is called a primer & synthesized
by primase

• Primase start a complementary RNA chain

• and adds RNA nucleotides using the parental DNA template strand

• Completed primer (5-10 nucleotides long) is therefore paired with the DNA
template strand

• The new DNA strand will start from 3’ end of the RNA primer
 SYNTHESIS OF TWO DAUGHTER CELLS

➢ DNA polymerase catalyzes the synthesis of new DNA strand

• by adding nucleotides to the 3’ end of the pre-existing chain (RNA primer)

 SYNTHESIS OF TWO DAUGHTER CELLS

➢ In E.coli several DNA Polymerases,

➢ Two play a major role in DNA replication

• DNA Polymerase III

• DNA Polymerase I
 SYNTHESIS OF TWO DAUGHTER CELLS

➢ Two ends of a DNA strand are different and the two strands of DNA are
antiparallel
• oriented in opposite direction to each other

• Therefore the two new strands formed during DNA replication must also
be antiparallel to their template strands
 SYNTHESIS OF TWO DAUGHTER CELLS

➢ The antiparallel arrangement of the double helix along with the limitations on the
function of DNA polymerase has effect on how replication occurs

• Because of their structure DNA Polymerase III can add nucleotides only to the
3’ end of a primer, never to the 5’ end

• As a result, a new DNA strand can elongate only in the 5’-3’ direction
 SYNTHESIZING THE LEADING STRAND

➢ Along one template strand, DNA Polymerase III

synthesize a complementary strand continuously
in the mandatory direction 5’-3’

• the Leading strand

• New DNA strand synthesized continuously

towards replication fork (5’-3’)

• Only 1 primer is required for DNA Polymerase

III to synthesize the leading strand
 SYNTHESIZING THE LEADING STRAND

1. After an RNA primer is made,

2. DNA polymerase lll adds DNA nucleotides to the

RNA primer,

3. Continue adding DNA nucleotides to the new

strand complementary to the parent strand
 SYNTHESIZING THE LAGGING STRAND

➢ To synthesize the other DNA strand in the mandatory 5-3 direction

• DNA Polymerase III must work along the other template strand in the direction
away from the replication fork

• Lagging strand
 SYNTHESIZING THE LAGGING STRAND

➢ Lagging strand is synthesized discontinuously as a series of segments

• The segments are called Okazaki fragments

SYNTHESIZING THE LAGGING STRAND
SUMMARY OF DNA REPLICATION IN IN E.COLI
 VIDEO

• https://www.youtube.com/watch?v=TNKWgcFPHqw
 MATCH COLUMN A & B
A B
1. Template strand A. The 5’-3’ strand that is synthesized continuously in
DNA replication
2. RNA splicing B. 3’-5’ DNA strand used for RNA synthesis
(transcription)
3. β-subunit of DNA polymerase III C. Enzyme responsible for synthesizing RNA primer
for DNA replication

4. DNA polymerase III D. Forms a sliding clamp that surrounds DNA strands

5. Leading strand E. Post-transcriptional modifications in Eukaryotes

primary mRNA
6. Okazaki fragments F. Main polymerization enzyme used in DNA
replication
7. Replication folk G. Discontinuously synthesized DNA fragments in the
lagging strand
8. Primase H. Segment of the DNA opened by Helicase
PROOF READING AND
REPAIRING DNA
 PROOF READING AND REPAIRING DNA

➢ Pairing errors between incoming nucleotides and those on the template occurs
during DNA replications (base pairing)

➢ However, the error rates in the completed DNA molecule are lower

• Because during DNA replication many DNA polymerases proofread each

nucleotide against its template as soon as they are covalently bonded to the
growing strand
 PROOF READING AND REPAIRING DNA
➢ If there is incorrectly paired nucleotide, Polymerase removes the incorrect paired
and replaces it with the correct nucleotide
• Similar to fixing texting error by deleting the wrong letter and then enter the
correct one

➢ Sometimes mismatched nucleotides escape the proofreading by a DNA

polymerase,
• As a result, specific enzymes remove and replaced incorrect paired
nucleotides
• & this process is called Mismatch repair
 PROOF READING AND REPAIRING DNA

➢ Incorrectly paired or altered nucleotides can also occur after replication

• DNA molecules may undergo chemical changes under normal cellular conditions
• However, these changes are normally corrected before they become permanent
change (mutation)

• Each cell continuously monitor and repair its genetic material

 PROOF READING AND REPAIRING DNA

➢ Because repairing of damaged DNA is important in the survival of an organism

• several DNA repairing enzymes have evolved

➢ During repairing mechanism a segment of the strand containing the damaged is cut
out by a nuclease enzyme

• The resulting gap is filled in with nucleotides using the undamaged strand as a
template
 PROOF READING AND REPAIRING DNA

➢ Enzymes responsible in filling the gaps are

called DNA polymerases and Ligase

➢ There are several such DNA repair system and

one is called nucleotide excision repair
• Removes and then correctly replaces a
damaged segment of DNA using the
undamaged strand as a guide
 PROOF READING AND REPAIRING DNA

➢ E.g: An important function of the DNA repair

enzymes in our skin cells is to repair genetic
damage caused by the UV rays of sunlight
 REPLICATING THE ENDS OF DNA MOLECULE
➢ For linear DNA, such as DNA of eukaryotic chromosomes, the replication process
cannot complete the 5’ ends of daughter DNA strands
• because there is no 3’ end of a pre-existing chain (polynucleotide) for DNA
polymerase to add to

• As a result, repeated rounds of replication produces shorter DNA molecules

with uneven ends

➢ Most prokaryotes have a circular chromosome, with no ends

• The shortening of DNA does not occur
REPLICATION PROBLEM IN EUKARYOTIC ENDS

➢ The replication of linear chromosome ends

poses a problem,

• After the leading strand has been completely

extended to the last nucleotide

• The lagging strand has a single strand DNA

gap that must be primed and filled in
REPLICATION PROBLEM IN EUKARYOTIC ENDS

➢ The problem arises when the RNA primer at

the ends removed for replacement with DNA

➢ RNA primer cannot be replaced with DNA

• because there is no 3’ end for nucleotide

addition

• DNA Polymerase III can add

nucleotides only to
the 3’ end of a primer, never to the 5’ end
REPLICATION PROBLEM IN EUKARYOTIC ENDS

➢ As a result, the genetic information in the gap

will be lost in the next round of replication

➢ And repeated/next rounds of replication will

produce shorter DNA molecules with uneven
ends
What protects the genes of linear eukaryotic chromosomes
from being lost during repeated rounds of DNA replication?
➢ Eukaryotic chromosomal DNA have nucleotide sequences called telomeres at their
ends

• Telomeres do not contain genes,

• Instead the DNA consists of multiple repetitions of one short nucleotide sequence
 What protects the genes of linear eukaryotic chromosomes
from being lost during repeated rounds of DNA replication?

➢ E.g. in each human telomere, the six nucleotides sequence TTAGGG is repeated
between 100 and 1,000 times