DNA

 Replica,on  
 DNA structure  
• History  
• DNA  is  polynucleo,de;  nucleo2des  are  
composed  of  a  phosphate,  a  sugar  and  a  
nitrogen  containing  base.  
 Structure of DNA  
• James  Watson  &  Francis  
Crick  determined  the  
structure  of  DNA  in  
1953.  
 Double Helix
• Each  side  of  the  double  helix  runs  in  opposite  
(an,-­‐parallel)  direc2ons.  
• The  beauty  of  this  structure  is  that  it  can  unzip  
down  the  middle  and  each  side  can  serve  as  a  
paOern  or  template  for  the  other  side.    
 Replication
• Replica  “copy”.  
• DNA  making  copies  of  itself,  we  call  it  DNA  
replica,on  .  
• DNA replication is the process in which the DNA
within the cell makes an exact copy of itself.
• It is a biological process that occurs in all living
organisms and copied their DNA.
• basis for biological inheritance.
 Why DNA replicate itself?

• To  reproduce,  a  cell  must  copy  and  

transmit  its  gene2c  informa2on  (DNA)  to  
all  of  its  progeny.  To  do  so,  DNA  
replicates.  
• DNA  carries  informa2on  for  making  all  of  
the  cell’s  protein.  
 
 Replication in Different Cells

• Different  types  of  cells  replicated  their  DNA  at            

different  rates.    
• Hair  cells,  finger  nails,  bone  marrow  cells  constantly  
divide.  
• Cells  of  brain,  heart  and  muscles  cells  go  through  
several  rounds  of  cell  division  and  stop.  
• Skin  cells  and  liver  cells,  stop  dividing,  but  can  be  
induced  to  divide  to  repair  injury.    
 Where replication occur?

• In  prokaryotes,  DNA  replica2on  occurs  in  the  

cytoplasm.  
• In  eukaryotes,  in  the  nucleus.  
 
 DNA  replica,on  models  
• Conserva,ve-­‐  would  leave  
the  original  strand  intact  
and  copy  it.  
• Dispersive-­‐would  produce  
two  DNA  molecule  with  
sec2ons  of  both  old  and  
new  along  each  strand.  
• Semi  conserva,ve  -­‐would  
produce  DNA  molecule  
with  both  one  old  strand  
and  one  new  strand  
 Requirements
of replication
• DNA  template.  
• Free  3’-­‐OH  group.  
• Proteins/enzymes  of  DNA  replica2on  
 DNA template
• Template  strand,  that  is  to  be  copied.  
• Each  old  strand  act  as  a  template.  
 
Free 3’-OH group
 Proteins and Enzymes of
Replication
1. DNA  Helicases    
2. DNA  Polymerase    
3. DNA  single-­‐stranded  binding  proteins  
4. DNA  Gyrase    
5. DNA  Primase  
6. DNA  Ligase      
 DNA Helicase
 DNA  Helicases  -­‐  These  proteins  bind  to  the  double  
stranded  DNA  and  s2mulate  the  separa2on  of  the  
two  strands.  
Role  in    
• Transla2on  
• Transcrip2on  
• RNA  slicing  
• RNA  Transport  
• RNA  Degrada2on  
 
 DNA  polymerase  
• a  cellular  or  viral  polymerase  enzyme  that  
synthesizes  DNA  molecules  from  
their  nucleo2de  building  blocks.    
• func2on  in  pairs  while  copying  one  double-­‐
stranded  DNA  molecule  into  two  double-­‐
stranded  DNAs.  
Func,on  of  DNA  polymerase  
 Synthesis  
       It  is  important  to  note  that  
the  direc,onality  of  the  newly  forming  strand  
(the  daughter  strand)  is  opposite  to  the  
direc2on  in  which  DNA  polymerase  moves  
along  the  template  strand.  Since  DNA  
polymerase  requires  a  free  3'  OH  group  for  
ini2a2on  of  synthesis.  
 Types  of  DNA  polymerases  
• POL I
• POL II
• POL III
• POL I V
• POL V
 Applica2ons  of  DNA  polymerase  
• DNA  polymerases  also  play  key  roles  in  other  
processes  within  cells,  including  DNA  
repair  gene2c,  recombina2on  reverse  
transcrip2on  and  the  genera2on  of  an2body  
diversity.    
  
DNA single-stranded binding
proteins  
• as  a  tetramer  
• stabilize  the  single-­‐stranded  structure.  
• Replica2on  is  100  2mes  faster  when  these  
proteins  are  aOached  to  the  single-­‐stranded  
DNA.  
• DNA  Gyrase  -­‐  This  enzyme  catalyzes  the  
forma2on  of  nega2ve  supercoils  that  is  thought  
to  aid  with  the  unwinding  process.  
• DNA  Primase  -­‐  The  requirement  for  a  free  3'  
hydroxyl  group  is  fulfilled  by  the  RNA  primers  
that  are  synthesized  at  the  ini2a2on  sites  by  
these  enzymes.  
• DNA Ligase - forms  a  covalent  phosphodiester  
linkage  between  3'-­‐hydroxyl  and  5'-­‐phosphate  
groups.  
 Direction of replication
• It  replicates  from  3’  to  5’  of  the  template  
strand.  
• From  5’  to  3’  of  the  newly  growing  strand.  
 Replication fork
• DNA  does  not  unzip  en2rely.  It  unzips  in  a  
small  area  called  a  replica,on  fork,  which  
then  moves  down  the  en2re  length  of  the  
molecule.  
 Leading strand
• Leading  strand  synthesis  is  con2nuous.  
•  From  3’  to  5’  of  the  template.  
 Lagging strand
• Lagging  strand  synthesis  is  discon2nuous.  
•  Okazaki  fragments.  
• Ligase  joins  discon2nuous  fragments.  
 Steps of replication
• Ini2a2on  
• Elonga2on    
• Termina2on    
• Uncoiling Of Strand
• Formation Of Leading And Lagging Strand
• Formation Of Replication Fork.
• Addition Of Nucleotides To proceed Both
strands.
• Involvment of different enzymes
(DNA polymerases,DNA gyrase,DNA ligase,
telomerases)
• End point is termination.
 Initiation
• Helicase  unwinds  and  
separate  DNA  strand.  
• When  DNA  double  
helix  unwinds,  it  
resembles  a  ladder.  
• Bacteria have a single
origin or a single site of
replication per circular
chromosome.
•Eukaryotes have multiple
origins of replication.
•According to a research
there are up to 100,000
origins of replication
initiation in Humans
because there is a larger
store of genetic material.
 Elongation
• RNA  primase  lays  down  primers.  
• Replica2on  starts  at  primer  and  lays  down  
nucleo2des  5’  to  3’.  
• Leading  strand  goes  con2nuously,  lagging  
strand  goes  discon2nuously.  
 Replica2on  Fork  
•Structure that is formed Within nucleus during
DNA Replication.

•Created by Helicase which break Hydrogen

bond.

•Resulting intwo branching structure each one

made upof single strand of DNA

•These Two Strands Serve As Template For

Leading And Lagging Strands
 Leading  strand  
• Leading strand is a template strand of
DNA double helix in which replication fork
moves along 3’ to 5’ direction.

• Newly synthesized complimentary strand

will moved to 5’ to 3’ direction.

• Polymerase reads DNA and add

nucleotide to leading strand.
 Leading strand
• Leading  strand  synthesis  is  con2nuous.  
•  From  3’  to  5’  of  the  template.  
  
Leading  strand   Lagging strand
 

• Synthesized in • Synthesized in
same direction as opposite direction
movement of than leading strand.
replication fork. • Moves along 5’ to 3’
• Moves along 3’ to direction.
5’ direction. • Synthesized in short
• Synthesized segments called
continuously. okazaki fragments.
 Okazaki fragments

¡ These fragments are short, newly

synthesized DNA fragments that are formed
on lagging template strand.
¡ Complementary to lagging strand forming
short double stranded DNA section
¡ they are between 1000 to 2000 nucleotide
long in E. coli and are between 100 to 200
nucleotides long in eukaryotes.  
 Termination  

• RNA  primer  is  removed.  Replaced  with  DNA  

nucleo2des.  
• DNA  ligase  joins  okazaki  fragments  with  
phosphodiester  bonds.  
• Helicase  rewinds  DNA  together.  
 Termination

• Two methods
1. a termination site sequence in the DNA.
2. a protein which binds to this sequence to
physically stop DNA replication proceeding.
(DNA replication terminus site-binding
protein, Ter protein).
 
 Comparison in eukaryotic and
bacterial replication
Eukaryotes Bacteria
• Circular DNA structure
• Linear DNA structure
• DNA replication is controlled • Not a well-defined cell cycle
within the context of the cell but instead continuously copy
cycle. As the cell grows and their DNA; during rapid
divides, it progresses growth, this can result in the
through stages in the cell
cycle; DNA replication concurrent occurrences of
occurs during the S phase multiple rounds of replication.
(synthesis phase). The In E. coli, the best-
progress of the eukaryotic characterized bacteria, DNA
cell through the cycle is
controlled by cell cycle replication is regulated
checkpoints through several mechanisms,
including: the hemimethylation
and sequestering of the origin
sequence, the ratio of ATP to