DNA REPLICATION
SIR P.T. SCIENCE COLLEGE, MODASA
[Link] MICROBIOLOGY SEM-04
ROLL NO-3519
NAME-KATARA MAULIKBHAI PRATAPBHAI
UNDER THE GUIDANCE OF KRISHNA MADAM
INTRODUCTION
• DEFINITION : Mechanism by which DNA makes a copy of itself.
• Occurs during S-PHASE of cell division and is the basis of
inheritance in all living organisms.
IMPORTANT PROPERTIES OF DNA
REPLICATION
• DNA replication is autocatalytic function of DNA.
• DNA → DNA (Replication) Autocatalytic.
• DNA→RNA (Transcription) Heterocatalytic.
• SEMICONSERVATIVE : DNA molecule composed of 2
antiparallel strands the base sequence of each parent strands
serves as template for synthesis of new complementary
strands. Therefore out of two newly formed DNA molecule one
is new and other is conserved from old strand.
• Semi-conservative model of DNA replication : Meselson Stahl
Experiment.
• Alternative methods:Conservative and dispersive method.
• DNA synthesis occurs in 5’→3’ direction using dNTP precursor.
• REPLICON : Unit of DNA in which replication starts from an
origin and proceeds called replicon.
• PROKARYOTIC REPLICON: Circular (Usually) In bacterial
genome, replication proceeds bidirectionally. Therefore they
form single replicon and are monorepliconic.
• EUKARYOTIC REPLICON: Contain multiple replication origins
on single chromosomes hence multirepliconic (40-100Kb in
size)
REQUIREMENTS FOR DNA REPLICATION
1. DNA tempelate →Parent DNA
2. Origin of Replication : 1. Unique sequence of DNA [Link]
shorts repeated sequences. [Link] multimeric origin
binding proteins (role in) assembling DNA Polymerase and
other replication enzymes at replication site. 4. AT rich
stretch that allows unbinding of DNA duplex as less energy is
required to melt AT than GC.
3. dNTPs(Deoxyribonucleoside 5’-phosphate) dATP, dTTP, dGTP,
dCTP(Sugar+N Base + 3 Phosphate)
4. Mg+2 ( optimizes DNA Polymerase activity)
ENZYMES INVOLVED 1. DNA HELICASE : Helicase opens up the
IN DNA DNA double helix at the replication fork
and provide single stranded tempelate.
REPLICATION 2. SINGLE STRANDED BINDING PROTEINS
(SSB PROTEIN) : Binds to both separated
strands inhibit re-annealing.
3. PRIMASE : Synthesizes a short RNA
primes(<15 nucleotide) to prime DNA chain
elongator.
4. TOPOISOMERASE : DNA topoisomerase in
nucleus that break a phosphodiester
bonds in a DNA strands function. 1-
Relaxation 2-Knotting/Unknotting 3-
Catenation 4-Decatenation.
TYPE 1 w protein from E. coli TYPE 2 DNA gyrase
5. DNA polymerase : DNA dependent DNA polymerase enzyme
that can synthesizes a new DNA strand on a DNA tempelate.
DNA POLYMERASE INVOLVED IN BACTERIAL GENOME REPLICATION
3'→5' 5’→3'
Enzyme Subunits Exonuclease Exonuclease Function
Activity Activity
DNA repair and
DNA Pol I 1 Yes Yes
gap filling
DNA Pol II 7 Yes No DNA repair
Main replicating
DNA Pol III At least 10 Yes No
genome
POLYMERASE ENZYME INVOLVED IN EUKARYOTIC GENOME REPLICATION
3’→ 5' Exonuclease 5’→ 3' Exonuclease
Enzyme Function
Activity Activity
Priming during
DNA Pol α No No
replication
DNA Pol β No No Base excision repair
Mitochondrial DNA
DNA Pol γ Yes No
replication
DNA Pol δ Yes No Lagging strand synthesis
DNA Pol ε Yes No Leading strand synthesis
DNA Pol κ - - Required for attachment
• DNA LIGASE : Catalyse the joining of ends of two DNA chain by
forming phosphodister bond between 3’-OH group at one end
of 5’ phosphate group at the end of another DNA.
• RNA PRIMER : As DNA polymerase cannot initiatiate new DNA
strands but can only elongate by adding dNTPs at 3’-OH free
group in preexisting DNA.
• But RNA polymerase (primer) can initiates RNA chain, so
during replication short RNA sequence called primers are
formed on both the tempelate strands.
MECHANISM OF DNA REPLICATION
1. INITIATION
2. ELONGATION
3. TERMINATION
INITIATION
1. Local separation of two DNA strands (denaturation) at a
specific DNA site called origin of replication (ori)
2. Loading of Helicase : DNA helicase first get bound to initiator
protein and then is loaded onto DNA. Uncoiling of DNA is
brought by DNA helicase that generate unpaired strands in
open complex.
3. Uncoiling of DNA create torque that is transmitted to other
unreplicated part of DNA which becomes supercoiled.
Topoisomerase enzyme gyrase brings about relaxation of
supercoiled DNA
4. Helicase move along DNA using energy from ATP hydrolysis to
separate the strands.
5. The separated strands are inhibited from the re-annealing by (SSB
PROTEIN) which binds to both strands.
6. Helicase opens up helix and binds primase to form primosome.
7. Primase get activated to synthesizes RNA primers that have free
3’-OH group to which new dNTPs can be added by DNA pol 3 .
8. DNA polymerase 3 catalyses the formation of phosphodister
bonds between 3’-OH at last dNMP and 5’-Phosphate of the
dNTPs with reales of 2 of 3 phosphate.
9. Parent DNA acts as a template for addition of dNTPs by DNA
polymerase 3 . If “A” on tempelate strands “T” will be added.
[Link] DNA Polymerase 3 remain bound to DNA and move along
it’s length till replication complete.
[Link] eukaryotes DNA pol subunits catalyzes the formation of
phosphodister bond in elongation.
ELONGATION
1. DNA polymerase catalyzes the step by step addition of dNTPs to
DNA chain.
2. The two new strands are of opposite polarity one is continuously
and called leading strands. Other is synthesized discontinously
called logging strand.
3. Lagging strand require many RNA primers. These short fragments
are called Okazaki fragments.
4. After 1000-2000 nucleotides of leading strand have been
replicated, the first round of discontinuous strand synthesizes on
lagging strand can begin by the same machinery enzymes.
5. Joinig of Okazaki fragments on lagging strands to make continues
strands require two enzymes DNA polymerase 1 and DNA LIGASE.
6. As each Okazaki fragments formation complete, the DNA
primers of the previous fragments is removed by 5’→3’
Exonuclease Activity of DNA polymerase 1 and continues the
synthesis of newer DNA fragments.
7. Fragments are joined into continues strands by DNA LIGASE.
TERMINATION
• All eukaryotes chromosomes end in telomere.
• At telomere region of each of the newly synthesized strands there is
RNA primers at 5’ end and DNA pol. Cannot fill gap after RNA primer
is removed.
• Eukaryotes have tandemly repeated sequence at the end of
chromosomes.
• The enzyme telomere is a ribonucleoprotein and it contains RNA
and protein. It act as a reverse transcriptase and resynthesizes DNA
using RNA tempelate.
• Telomere catalyzes the new telomere repeat using RNA tempelate.
The process is repeated and more telomere repeats are added to
produce a net longer chromosomes with a new 5’ gap to avoid net
strategy of chromosomes.
QUESTION
1. In which phase of cell division, DNA replication is seen?
2. What is DNA replace?
3. Which enzyme open up double helical structure ? from
where?
4. In which direction DNA polymerase add nucleotide?
5. Which experiment gave concept of semi-conservative DNA
replication?
6. ________________contain multiple replication origins on single
chromosomes hence multirepliconic (40-100Kb in size)
7. Uncoiling of DNA create torque that is transmitted to other
unreplicated part of DNA which becomes supercoiled.
__________________ enzyme gyrase brings about relaxation of
supercoiled DNA.
8. Lagging strand require many RNA primers. These short
fragments are called ______________ fragments.
9. Which of the following enzymes function is to DNA repair
and gap filling?
A. DNA POLYMERASE 1 B. DNA POLYMERASE 2 C. DNA POLYMERASE 3 D. ALL
OF THE ABOVE
REFERENCE
Prescott’s principles of microbiology
THANKS