Cell and Molecular Biology

BS 4th Semester M/E

Course Code: ZOOL-303 Credit hours: 3(3-0)

Dr. Lubna Rasool

Department of chemistry
University of Sahiwal
 Objective

• To provide understanding about DNA replication in cell

• To provide understanding about DNA for the ultimate

maintaining and growth of organism and cell

 Cell cycle

• A series of events that takes place

in a cell

• A cell spends most of its time in

interphase

• Mitosis produce daughter cells

……Continued
• The cell cycle is a 4-stage process consisting of Gap 1 (G1),

Synthesis, Gap 2 (G2) and mitosis.

• G1, S and G2, are known collectively as the interphase.

• G1 phase: Cell increases in size, Cellular contents duplicated

• S phase: DNA replication, Each of the 46 chromosomes (23

pairs) is replicated by the cell

 ….. Continued
• G2 phase: Cell grows more, Organelles and proteins develop in

preparation for cell division

• M phase: Mitosis followed by cytokinesis (cell separation)

• Formation of two identical daughter cells

• G0 phase: Cell is performing its function without actively

preparing to divide.
 Cell Division (mitosis)
• DNA synthesis before divide the cell

• Centromere remains uncopied where

sister chromatids attached

 DNA Replication
Duplication of the entire genome prior to cell division

Biological significance

• Extreme accuracy of DNA replication is necessary

• In eukaryotes , replication only occurs during the S phase of the

cell cycle.

• Replication rate in eukaryotes is slower resulting in a higher

fidelity/accuracy of replication in eukaryotes

 DNA Replication
Three possible models were proposed for DNA replication:

▪ Conservative model.

▪ Dispersive model

▪ Semiconservative
• Conservative model proposed both strands

of one copy would be entirely old DNA,

while the other copy would have both

strands of new DNA.

• Dispersive model was that dsDNA might

fragment, replicate dsDNA, and then reassemble,

creating a mosaic of old and new dsDNA

regions in each new chromosome.

• Semiconservative model is that DNA

strands separate, and a complementary

strand is synthesized for each, so that

sibling chromatids have one old and one

new strand.
 Three models for the replication of DNA

Pet er J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Meselson-Stahl Experiment

▪ Meselson and Stahl (1958) grew E. coli in a heavy (not

radioactive) isotope of nitrogen, 15N in the form of 15NH4Cl.

▪ 15N is heavier than 14N.

▪ DNA containing 15N is more dense than DNA with normal 14N,

and so can be separated by CsCl density gradient centrifugation.

Equilibrium centrifugation of DNA of different densities in a
cesium chloride density gradient
 Meselson-Stahl Experiment

▪ Labeled 15N E. coli were shifted the cells to medium containing

normal 14N.

▪ DNA was extracted from each sample and analyzed in CsCl

density gradients.
The Meselson-Stahl experiment, which showed that
DNA replicates semiconservatively
 Meselson-Stahl Experiment

▪ After one replication cycle in normal 14N medium, all DNA

had density intermediate between heavy and normal.

▪ After two replication cycles, there were two bands in the

density gradient, one at the intermediate position, and one at

the position for DNA containing entirely 14N.

 Results

1. Does not fit to conservative model and dispersive model

2. The semiconservative model fits the data very well.

 DNA Replication
• The DNA bases on each strand act as a

template to synthesize a complementary

strand

• A pairs with T

• G pairs with C

• The process is semiconservative

because each new double-stranded DNA

…… Continued
AT
GC
AT C G
GC T A
C G GC
T A
GC
AT A T
GC GC
A T C G C G
G C T A T A
C G GC GC
T A
G C
 DNA Polymerases

• DNA Replicating Enzymes

• In 1956, Arthur Kornberg and colleagues discovered DNA

poly I in Escherichia coli.

• Later awarded the Nobel Prize in Physiology or Medicine in

1959
…. Continued

• DNA poly II was discovered by Thomas Kornberg

▪ Malcolm E. Gefter in 1970 , the role of Pol I in E. coli DNA

replication

▪ DNA polymerase III (1971), DNA polymerase IV, and DNA

polymerase V isolation also reported

….. Continued

• DNA polymerase is an enzyme that synthesizes DNA molecules

from deoxyribonucleotides.

• Deoxynucleoside triphosphate + DNAn ⇌ diphosphate +

DNAn+1.

• It "reads" the existing DNA strands to create two new strands

that match the existing ones.

 ….. Continued
• DNA polymerase structure is highly conserved

• Only extend an existing DNA or RNA strand paired with a

template strand.

• A short fragment of RNA, called a primer, is required.

• DNA polymerases are highly accurate

• In addition, some DNA polymerases also have proofreading

ability.
DNA chain elongation catalyzed by DNA polymerase
 Rate of DNA replication

• Firstly, measured in phage-infected E. coli.

• During the period of exponential DNA increase at 37 °C, the

rate was 749 nucleotides per second.

 Basic rules of replication

1. Semi-conservative

2. Starts at the ‘origin’

3. Uni or bidirectional

4. Semi-discontinuous

5. Synthesis always in the 5-3’ direction

6. RNA primers required

Please study the topics from Campbell Biology

Thank you