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DNA & Histone
Mitosis (M) synthesis
S phase
takes place
only after DNA G1
synthesis (S). phase
Two gaps (G1
and G2) in
G2
time separate phase
the two M
phase
processes.
DNA Replication

???????????
 DNA Replication

Duplication of DNA

New DNA strands are synthesized by using the existing

(parental) strands as templates in the formation of new,
daughter strands complementary to the parental strands
Both the strands of parental DNA serve as templates and
synthesis occurs simultaneously on both the strands

High degree of fidelity

Always 5¶ 3¶ (template is read in ????)
DNA replication is bidirectional
 DNA Replication
Semi conservative

Each of the parental

strands serves as a
template for a daughter
strand
 DNA Replication
Requirements:
Templates
Deoxy ribonucleotides (dATP, dGTP,dCTP,TTP)
Enzymes to polymerize
Unwinding/separation of the strands
Maintenance of single stranded status
Primer
Editing (Proof Reading)
 Prokaryotic DNA Replication
1. Origin of replication (oriC locus)
 Prokaryotic DNA Replication

1. Origin of replication (oriC locus)

a. Rich in AT base pairs ???
b. In E Coli, the oriC is bound by the protein dnaA
which brings local denaturation and unwinding of
adjacent AT region of DNA (³melting´)
c. ATP requiring process
 Prokaryotic DNA Replication
2. Unwinding of DNA
a.The interaction of proteins with ori defines the start site
of replication and provides a short region of ss DNA
essential for initiation of synthesis the nascent DNA
strand
b. Requires the formation of a number of protein-protein
and protein-DNA interaction
c. DNA helicase using ATP hydrolysis allows progressive
unwinding of DNA. In E.coli it is a complex of
dnaB helicase and dnaC protein
d. Maintainence of unwinding state and free from
nucleases attack--
Single-stranded DNA binding proteins (SSBs)
 Prokaryotic DNA Replication

2. Unwinding of DNA (contd..)

DNA + Helicase complex + SSBs = ³prepriming complex´

 Prokaryotic DNA Replication
3. Formation of replication fork

Has 4 components that form in the following sequence

 Prokaryotic DNA Replication
3. Formation of replication fork (contd..)

i. The DNA helicase unwinds a short segment of the

parental duplex DNA
ii. A primase initiates synthesis of an RNA molecule
that is essential for priming DNA synthesis and is
complementary to the unwound template strands??
(primer is about 10-200 ribonucleotides)

Primase + prepriming complex = Primosomes

(³The mobile complex´)
 Prokaryotic DNA Replication
3. Formation of replication fork (contd..)
iii. The primer, still base-paired to its complementary
DNA strand, is then elongated by a DNA polymerase²
new daughter strand
 Prokaryotic DNA Replication

3. Formation of replication fork (contd..)

iv. SSBs bind to a ssDNA and prevent premature
reannealing of ssDNA to form dsDNA
 Prokaryotic DNA Replication
Complications in the replication
1. Antiparallel
2. DNA ploymerase can add nucleotides to the
growing new strands only in the 5¶ 3¶ direction
Synthesizing a new primer every few hundred bases
³OKAZAKI FRAGMENTS´ (RNA primer and a DNA fragment
in the lagging strand)
 Prokaryotic DNA Replication

Result: Leading strand (forward strand) (continuous) Moves towards the replication fork
Lagging strand (retrograde strand) (discontinuous) Moves away from the fork
 Prokaryotic DNA Replication
DNA Polymerases
Polymerises deoxyribonucleotides
Synthesis DNA in the 5¶ 3¶
(No polymerase in 3¶ 5¶)
Template-directed enzymes
Needs RNA primer
Different types of DNA polymerases
Share important 3 properties
1. Chain Elongation
2. Processivity (an expression of the # of nucleotide added to the
nascent chain before the polymerase disengages
from the template)
2 identical ȕ subunit of Pol III forms a ³clamp "around the
template
3. Proof Reading (Identifies copying errors and corrects them)
 Prokaryotic DNA Replication
Functions of DNA polymerases
DNA polymerases of E. coli

pol I pol II pol III (core)

Polymerization: 5¶ to 3¶ yes yes yes
Proofreading exonuclease: 3¶ to 5¶ yes yes yes
Repair exonuclease: 5¶ to 3¶ yes no no

DNA polymerase III is the main replicating enzyme

DNA polymerase I has a role in replication to fill gaps and excise
primers on the lagging strand, and it is also a repair enzyme
and is used in making recombinant DNA molecules
DNA polymerase II participates in DNA repair
 Prokaryotic DNA Replication
Major Steps In Replication
1. Initiation
2. Elongation
3.Termination
INITIATION
1. Origin of replication (oriC): dnaA
2. Unwinding of DNA: Helicases- dnaB and dnaC
SSB
3. Synthesis of RNA primer: Primase(formation of DNA-RNA hybrid)
4. Entry of first dNTP:DNA polymerase III
 Prokaryotic DNA Replication
jONGATION
1. DNA Polymerase III (5¶ 3¶ polymerase activity)
2. The template dictates which dNTP is complementary and by
hydrogen bonding holds it in a place while the 3¶ OH group
of the growing strand attacks and incorporates the new
nucleotide into the polymer (For picture see the slide #21)
3. ȕ subunit of Pol III forming a ³clamp "around the template
4. Only a small stretch of the template duplex is single-
stranded at any given time.
5. Formation of replication fork and leading and lagging
strand.(Semi discontinuous DNA synthesis)
6. Formation and deformation of super coils (for details ref slide #
22-24)
7. Proof Reading: DNA Polymerase III (3¶ 5¶ exonuclease
activity)
8. Bidirectional (For picture see the slide #25)
9. Reformation of the helix
Prokaryotic DNA Replication

Initiation

Elongation
 Prokaryotic DNA Replication
jONGATION 
The progression of fork requires continuous unwinding of DNA

Severe torsional stress into the duplex ahead of the fork

³DNA Topoisomerase´

1. Cleaves 1 (Type I) or both (Type II) strands of DNA

(³transient nick´)
2. Unwinding of broken end around the intact strand
3. Resealing
 Prokaryotic DNA Replication
jONGATION 
2 Types of topoisomerases
Type I
- Cleaves 1 strand
- No ATP involvement
Type II
- Cleaves both the strands
- Needs ATP
eg: DNA gyrase
- Bacteria and plants
- Neutralises positive super coils by introducing
negative super coils

Both the types are important in replication, transcription and

recombination
 Prokaryotic DNA Replication
jONGATION 

Drugs targeting Topoisomerase

‡ The bacterial topoisomerase II (often called DNA gyrase) is the
target of several antibiotics
± Y
  blocks the binding of ATP to gyrase.
± Y   a

   interfere with the breakage
and rejoining of DNA chains ´ widely used to treat urinary tract
and other infections.
‡ r
  an antitumor agent, inhibits human
topoisomerase I by stabilizing the form of the enzyme covalently
linked to DNA
‡ 
 anticancer agent, inhibits human topoisomerase II
 Prokaryotic DNA Replication
jONGATION 
 Prokaryotic DNA Replication
TjINATION   jONGATION 

?????????
 Prokaryotic DNA Replication
TjINATION 
1. Removal of primers: Pol I (5¶ 3¶ exonuclease activity)
2. Replacement of them by DNA: Pol I (5¶ 3¶ polymerase activity)
3. Proof Reading: Pol I (3¶ 5¶ exonuclease activity)
4. Ligation: DNA Ligase

The final phosphodiester linkage between 5¶ phosphate group

synthesized by Pol III and 3¶ OH group made by Pol I is catalyse
by DNA ligase and is ATP-dependent

The bacterial DNA molecule circularizes at the end of the

DNA ligase
Prokaryotic DNA Replication
 Eukaryotic DNA Replication
Essentially similar to prokaryotic replication
- Bidirectional
- RNA primers
- Leading and lagging strand

Bacterial genome is about 6X106 bp

Replication is completed in ~ 30 min
Replication rate is ~ 3X105 bp/min

Eukaryotic genome is about 3X109 bp

If the replication rate is 3X105 bp/min from a single Ori, then replication
would be for over 150 hrs!!!!

Solution????
 Eukaryotic DNA Replication
1. Bidirectional
2. Multiple origins in each chromosome

generates ³Replication Bubbles´

 Eukaryotic DNA Replication
Eukaryotic DNA polymerases
Polymerase Function Proof reading

Į Contains -
primase;initiates
primase ;initiates
replication
ȕ Repair -

Ȗ Replicates +
mitochondrial DNA
į Elongates +

İ Repair +
 Eukaryotic DNA Replication
DNA polymerase Į = RNA polymerase (primase)!!!!!!!!
+
DNA polymerase

DNA polymerase į = Elongation

Topoisomerase I (check prokaryotic part) (is it a DNA ligase?

and or endonuclease?
 Eukaryotic DNA Replication

PCNA (Proliferating Cell Nuclear Antigen)

- Acts like ȕ subunit of DNA polymerase III ????
- Binds to DNA polymerase į
- PCNA method is used as ³Mitotic Index´
 Eukaryotic DNA Replication
Telomeres and Telomerase
 Eukaryotic DNA Replication
Telomer is a region of repetitive DNA sequence at the end of a
Chromosome (AGGGTT)
 Eukaryotic DNA Replication
- Chromosome/DNA can not replicate right to the tip
-Limited somatic cell division

Sequences are lost in every replicative phase

Critical level

cell division stops

Ageing!!!!!!! (  

If sequences are protected from loss

Stem cells, germ
cells, Cancer cells
Immortal !!!
 Eukaryotic DNA Replication
5¶ 3¶
3¶ 5¶

Primer on lagging strand

5¶ 3¶
3¶ 5¶

-No Polymerase to fill the gap

-One long and one short strand
-Protect the single strand???
-Continues, reaches a critical level
-Cell ages !!

Solution in stem cells, germ cells???

 Eukaryotic DNA Replication
Loss is inevitable
Loss is replenished!!!

5¶ 3¶
3¶ 5¶

Next replication
5¶ 3¶
3¶ 5¶
 Eukaryotic DNA Replication

Telomerase is a unique reverse transcriptase

- A cellular reverse transcriptase
- Contains intrinsic RNA component (ribonucleoprotein)
- Specializes in synthesizing multiple repeats
 Eukaryotic DNA Replication
  T  

‡ Retroviruses (HIV) genome is ss RNA

‡ After infection of host cell, (
a
(

a 
a
using the RNA as template synthesises DNA ´ reverse
transcription
‡ ma

a
a ( ´   a  
a
´ responsible for the development of drug resistance and
difficulties in developing a vaccine
 Eukaryotic DNA Replication
N   


 a  

‡ Nucleoside analogs formed by
 a

   

± Didanosine ´ 2¶3¶ dideoxyinosine
± Cytarabine ´ cytosine arabinoside
± Vidarabine ´ adenine arabinoside
± Zidovudine
‡ prevent elongation of DNA slowing division of rapidly
growing cells and viruses
 Eukaryotic DNA Replication

 T 
‡ Alkylating agents ´ Ex: 
 
  
function by
± cross-linking of bases in the DNA
± induce mispairing of nucleotides
‡ act upon DNA at all stages of the cell cycle
‡ Anthracyclines ´ Ex : | 
± inhibit the actions of topoisomerase II
‡ 
 ´ inhibition of topoisomerase II
‡ r
  ´ inhibit the action of topoisomerase I
‡ anticancer drugs that interfere with nucleotide metabolism ´
antimetabolites
‡ Any other ???????????