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					DNA REPLICATION
 Dept of Biochemistry
       BPKIHS


                        1
         STEPS INVOLVED in DNA
             REPLICATION
1.   Identification of the origins of replication
2.   Unwinding (denaturation) of dsDNA to
     provide an ssDNA template
3.   Formation of the replication fork;
     synthesis of RNA primer
4.   Initiation of DNA synthesis and
     elongation
5.   Formation of replication bubbles with
     ligation of the newly synthesized DNA
     segments
6.   Reconstitution of chromatin structutre   2
I. General Features of Replication

  A. Semi-Conservative
  B. Origin of replication
  C. Bidirectional
  D. Semi-Discontinuous

II. Identifying Proteins and Enzymes of
   Replication

III. Detailed Examination of the Mechanism
   of Replication

   A. Initiation
   B. Priming
   C. Elongation
   D. Proofreading and Termination        3
                                  Q. During which phase
DNA replication is the process       of the cell cycle
where an entire double-stranded
                                    does DNA replicate?
DNA is copied to produce a
second, identical DNA double
helix.

Meselson and Stahl concluded
that the mechanism of DNA
replication is the
semiconservative model.

Each strand of DNA acts as a
template for the synthesis of a
new strand.



                                                     4
  Possible Models of DNA Replication
Conservative- would
leave the original strand
intact and copy it.

Dispersive-would
produce two DNA
molecule with sections of
both old and new along
each strand.

Semiconservative –
would produce DNA
molecule with both one
old strand and one new             5
strand.
            Initiation of Replication
   Initiation starts at a site called origin of replication.
   The origin of replication in E. coli is termed oriC
     – origin of Chromosomal replication
   Important DNA sequences in oriC
     – AT-rich region
     – DnaA boxes: 20-50 monomers binds with the site
       of origin causing the double stranded DNA to
       separate
   The ds DNA is unwound by Helicase.
   This generates positive supercoiling ahead of each
    replication fork


                                                                6
              Initiation of Replication
   DNA gyrase: relieves tension from the unwinding of the DNA
    strands during bacterial replication. It cuts nicks in both strands of
    DNA, allowing them to swivel around one another and then
    resealing the cut strands

   The single stranded binding proteins (SSBs) bind to the exposed
    bases to prevent them from annealing.
     Then short (5 to 50 nucleotides) RNA primers are synthesized
     by primase

   Primase is an RNA polymerase that makes the RNA primer.
     –These short RNA strands start, or prime, DNA synthesis

   DNA polymerases can synthesize DNA only in the 5’ to 3’ direction
    and cannot initiate DNA synthesis

    DNA synthesis is semidiscontinuous and bidirectional
    On the leading strand the DNA synthesis is continuous
    On the lagging strand the DNA synthesis is discontinuous
                                                                     7
     Building Complimentary Strands
   In prokaryotes, there are 3
    enzymes known to function in
    replication & repair
     – DNA polymerase I, II & III

   In eukaryotes,
    there are 5 enzymes
    known to function
    in replication & repair
    DNA pol α, β, γ, δ,ε




                                    8
9
Building Complimentary Strands
         in prokaryotes
   DNA polymerase III builds the complimentary strand of DNA
     – It only functions under certain conditions

   DNA polymerase III adds complimentary nucleotides
    (deoxyribonucleoside triphosphates) in the 5’ to 3’ direction,
    using RNA primers as starting points
     – The segments are called Okazaki fragments




                                                                10
       Building Complimentary
       Strands in prokaryotes
   RNA primers are synthesized by primase and are
    temporary
   The leading strand (uses 3’-5’ template) is
    synthesized continuously
   The lagging strand (uses 5’-3’ template) is
    synthesized discontinuously in short fragments




                                                11
       Building Complimentary
       Strands in prokaryotes
   DNA ligase joins the Okazaki fragments into one
    strand on the lagging strand of DNA through the
    formation of a phosphodiester bond.




                                                  12
     Building Complimentary
     Strands in prokaryotes
 DNA polymerase I removes the RNA primers from
the leading strand and fragments from the lagging
strand and replaces them with the appropriate
deoxyribonucleotides.




                                               13
Replication fork and DNA
        synthesis




                           14
        Building Complimentary
        Strands in prokaryotes
   As the 2 new strands of DNA are synthesized, 2
    double stranded DNA molecules are produced that
    automatically twist into a helix.




                                                  15
FACTORS NEEDED FOR PROPAGATION
     OF REPLICATION FORKS
1. DNA HELICASES
 to separate the strand
2. GYRASE (Topoisomerases)
 unwind the supercoil
3. Single strand binding protein
   (SSBP)
  activity of helicase
 keep two strand separate
 protect DNA from nuclease degradation
 release after replication          16
         FACTORS NEEDED FOR
           PROPAGATION ….
4.PRIMOSOME
       Complex Protein (contains)
   primase
   helicase dna B
   dna C
   protein n, ni, nii and i
   act as primer for DNA synthesis
   removed by polymerase I
5. DNA SYNTHESIS CATALYZED BY DNA POLYMERASE III
   New strand synth is catalyzed by DNA poly III.
   5'  3' direction.
   Antiparallel to parent template.
   dNTPs are added one after another to 3'.
   Newly synthesized strand nucleotide is complementary to parent
    DNA strand.


                                                             17
             PROOF READING

         of replication is important.
 Fidelity

 DNA Polymerase III – beside synth also
  perform proof reading activity.
 Check during synthesis.

 Correction




                                      18
            DNA LIGASE
 It catalyses the formation of
  phosphodiester linkage between small
  fragment of DNA.
TERMINATION
 Termination utilization sub (Tus)
  protein binds to termination
  sequence  prevent helicase dna B
  protein.
 Unwinding of DNA helix is stopped.

                                     19
     EUKARYOTIC REPLICATION
 Multiple origin site.
 Multiple replication bubbles.
 Enzymes – 5 types
 DNA polymerase - , , ,  and 
 DNA Poly  = Synth of RNA primer (both
  strand)
 Responsible for initiation
 Poly  = Repair of DNA
 Poly  = Replication of mitochondrial DNA
 Poly  = Replication of leading & lagging strand
 Proof reading
 Poly  = Lagging strand
                                               20

				
DOCUMENT INFO
Description: DNA Replication,Translation and transcription Recombinant DNA and new technology PCR,Iron metabolism,Hemoglobin synthesis,RFLP,Protein synthesis,Protein Targeting,Genetic Code,DNA mutation,Gene Amplification,Immunoglobulin,Plasma Protein,Albumin,Lac operon model,Gene regulation.DNA repair,Antigen Processing