REPLICATION

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REPLICATION M.Prasad Naidu MSc
Medical Biochemistry, Ph.D,.
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Watson and crick
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• Introduction
• Besides maintaining the integrity of DNA
  sequences by DNA repair, all organisms must
  duplicate their DNA accurately before every cell
  division.
• DNA replication occurs at polymerization rates of
  about 500 nucleotides per second in bacteria and
  about 50 nucleotides per second in mammals.
• Clearly, the proteins that catalyze this process
  must be both accurate and fast.
• Speed and accuracy are achieved by means of a
  multienzyme complex that guides the process and
  constitutes an elaborate "replication machine."

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• Replication occurs in 5 to 3 direction only.
• Replication is simultaneous on both strands.
• Replication is bidirectional.
• Replication obeys base pair rule
• Replication results in 2 daughter DNA strands.
• Each daughter DNA strand has one
• parent strand and one complementary
• strand synthesized newly. Hence this
• Replication is semi-conservative.
• Held by phospho-di-ester bonds
• and Hydrogen bonds

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CELL - CYCLE

• Cell cycle is a sequence of events that occur in
  a cell during cell division.
• It results in formation of 2 identical daughter
  cells.
• Duration of cell cycle varies from cell to cell.
• It occurs in 4 phases
• G1 PHASE gap-1
• S PHASE synthetic
• G2 PHASE gap-2
• M PHASE mitotic

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G0
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Cell- cycle

• G1 phase Preparative phase for DNA
  synthesis. All cellular components replicate
  except DNA . Cell size
• increases. Any damage to DNA is detected.
• S phase DNA replication takes place.
• G2 phase Prepares for cell division and spindle
  
• formation. Any damage to
  DNA is detected.
• M phase Cell undergoes cell division . It
  includes prophase ,metaphase, anaphase ,and
  telophase.
• After mitosis cell may continue cycle by
  re-entering into G1 or enter G0 and remain
  dormant or leads to cell
• death

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Models for DNA REPLICATION

• These are many hypothesis to explain the process
  of replication. They are
• Conservative model
• Semi conservative model
• Dispersive model

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Semi conservative model of replication
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Replication is Semiconservative
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Semi conservative model of replication
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DNA-Replication

• Requirements
• 1.Deoxyribonucleotides dATP, dGTP, dCTP, dTTP
  
• 2.Template DNA strand parent strand
• 3.RNA primer
• 4.Enzymes DNA polymerase
• Primase
• Helicase
• DNA Ligase
• Topo-isomerases
  
• Single Strand Binding
  Proteins.

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• Single strand binding protein (SSBP )
• Binds to ssDNA
• Has two function
• 1. prevents reannealing , thus providing
  ss template
• required by polymerases
• 2. protects ssDNA from nuclease activity
• Show cooperative binding

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• Helicases
• Separate the ds DNA to ss DNA by dissolving the
  hydrogen bonds holding the two strands together
• These separates dsDNA at physiological
  temperature
• ATP dependent
• At least 9 helicases have been described in E
  coli
• Of which DNA binding protein A, B , C ( Dna A,
  Dna B, Dna C ) are most important
• Initial separation is by Dna A
• Continued further by Dna B ( major strand
  separating protein acts bidirectionally )
• Dna C is required for loading Dna B at site of
  replication

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Primase

• Primase is a specilised RNA polymerase
• It synthesis a short strech of RNA in 5
  3 direction on a template running in 3
  5 direction.
• An RNA primer, about 100-200 nucleotides long,
  is synthesized by the RNA primase.
• The RNA primer is removed by DANP, using
  exonuclease activity and is replaced with
  deoxyribo nucleotides by DNAP

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• DNA Ligases
• DNA ligases close nicks in the
  phosphodiester backbone of DNA.  Two of the most
  important biologically roles of DNA ligases are
• 1. Joining of Okazaki fragments during
  replication.
• 2. Completing short-patch DNA synthesis occurring
  in DNA repair process. 
• There are two classes of DNA ligases
• The first uses NAD as a cofactor and only found
  in bacteria. 
• The second uses ATP as a cofactor and found in
  eukaryotes, viruses and bacteriophages. 

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DNA Ligase Structure
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• DNA Ligase Mechanism
• The reaction occurs in three stages in all DNA
  ligases
• Formation of a covalent enzyme-AMP intermediate
  linked to a lysine side-chain in the enzyme.
• Transfer of the AMP nucleotide to the
  5-phosphate of the nicked DNA strand.
• Attack on the AMP-DNA bond by the 3-OH of the
  nicked DNA sealing the phosphate backbone and
  resealing AMP. 

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• SUPERCOILS
• As two strands unwind ,they result in the
  formation of positive supercoils ( super twists
  ) in the region of DNA ahead of replication fork.
• Accumulation of these supercoils interfere with
  further unwinding of ds DNA.
• This problem is solved by the enzyme
  Topoisomerases.
• These catalyze the interconvertion of topoisomers
  of DNA

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• Catalyze in a three step process
• 1. cleavage of one or both strands of
  DNA
• 2. passage of a segment of DNA through
  this break
• 3. resealing of the DNA
• Two types of topoisomerases are present
• DNA which different in the linking numer
• Linking number (Twist Wreth) 3 dimentional
• -type I topoisomerases
• -type II topoisomerases

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• Topoisomerases I
• Reversibly cut one strand of double helix
• Have both nuclease ( strand cutting ) ligase (
  strand resealing )
• Donot require ATP ,rather use the energy released
  by phosphodiester bond cleavage to reseal the
  nick
• Removes only negative super coils
• Ex bacteria

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• Topoisomerases II ( DNA gyrase )
• Heterodimer with 2 swivelase 2 ATPase subunits
• Swivelase subunit catalyzes trans esterification
  reaction that breaks reforms the
  phosphodiester backbone
• ATPase subunit hydrolyzes ATP to trigger
  conformational changes that allow a double helix
  to pass through the transient gap
• Possitive super coiled

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DNA polymerases

• These are the enzymes responsible for the
  polymerisation of deoxy ribo nucleosides,
  triphosphates on a DNA template strand to form a
  new complementary DNA strand.
• In prokaryotes based on site and conditions of
  action. They are divided into 3 types I II
  III.

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• Common properties
• All polymerases can synthesis a new strand of DNA
  in 5 to 3 direction. On a template strand which
  is running in 3to 5 direction.
• They also show Exo nuclease activity ( it
  cleaves the end terminals of DNA) in 3to 5
  direction.
• All DNA polymerases cannot initiate the process
  of replication on their own. This is the basic
  defect of DNAP synthesis of new strand .

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Comparison of prokaryotic eukaryotic DNA
polymerase
Prokaryotic Eukaryotic FUNCTION
l a Gap filling synthesis of lagging strand
ll e DNA proofreading repair
ß DNA repair
gamma Mitochondrial DNA synthesis
lll d leading strand synthesis
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Replication

• There are three phases of replication
  
• 1. Initiation
• 2. Elongation
• 3. Termination

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Steps in DNA-replication

• 1.Recognition of origin of replication and Un-
  winding of double stranded DNA
• 2.Formation of replication bubbles with 2
  replication forks for each replication bubble.
• 3.Initiation and elongation of DNA strand.
• 4.Termination and Reconstitution of chromatin
  structure.

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Unwinding of ds DNA

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Initiation of DNA-REPLICATION

• 1.Identification of the origins of replication.
• The origin of replication oriC locus rich in
• AT pairs is identified.
• A specific protein Dna A binds to the oriC
  and results in unwinding of ds DNA.
• Un winding of DNA results in formation of
  replication bubble with 2 replication forks.
• Ss binding proteins binds to DNA to each strand
• to prevent re-annealing of DNA.
• Helicases continues the process of un winding.
• Topoisomerases relieve the super coils formed
  during unwinding.

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Topo-isomerases
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DNA-replication

• 2.Fomation of replication fork
• replication fork has 4 components
• 1.helicase unwinds ds DNA
• 2.primase synthesizes RNA primer
• 3.DNApolymerasesynthesizes DNA
• 4.ss binding proteins stabilizes the
  strand

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2.Elongation of DNA

• Requires RNA primer, DNA template , DNAP
  enzyme
• and deoxyribonucleotides dATP,dGTP
  ,dCTP, dTTP
• DNA polymerase catalyze the stepwise addition
  of
• deoxyribonucleotides to 31 end of template
  strand and
• thus copies the information from the
  template DNA.
• DNAP requires RNA primer to start elongation.
• DNAP copies the information from DNA template

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2.Elongation of DNA

• 1.continous synthesis occurs towards the
  replication fork leading strand by DNA
  polymerase.
• 2.discontinuous synthesis occurs away from
  the replication fork in pieces called as okazaki
  fragments which are ligated by DNA ligase
  lagging strand. It requires multiple
  RNAprimers.

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• Okazaki fragments
• First demonstrated by Reiji Okazaki
• Short fragments of DNA present on the lagging
  strand resulted by retrograde synthesis.
• Okazaki fragments in human cells average about
  130 - 200 nucleotide in length
• In E coli they are about ten times this.

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Replication

• RNA primer is removed by DNAP with
• exonuclease activity. Again the gap is filled
  by DNAP. The two Okazaki pieces are later joined
  by DNA ligase.

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ROLE OF TELOMERS IN EUKARYOTIC REPLICATION

• A small portion of 31 end of parent strand is not
  replicated and length of chromosome reduces.
• Telomeres play a crucial role in eukaryotic
  replication.
• Telomeres contain the repeat sequence of
• TTAGGGn .
• They prevent the shorting of chromosome with each
  cell division by an enzyme telomerase.
• Telomerase enzyme synthesizes and maintains the
  telomeric DNA.
• Telomerase adds repeats to 31end of DNA

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3.Termination of DNA replication

• In prokaryotes the process of replication is
  terminated when the two replication forks moving
  in opposite directions from the origin meet.
• In E.coli replication of circular DNA takes about
  30 minutes.
• In eukaryotes replication is terminated when
  entire DNA is duplicated in S phase of cell cycle.

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Inhibitors of Replication

• 1.Inhibitors of DNA Prevents un-winding of DNA.
• E.g. actinomycin,
  mitomycin
• 2.Inhibitors of deoxy-ribonucleotides
• E.g.
  Anti-folates inhibits Purine\
• 
  Pyrimidine synthesis
• 3.Inhibitors of replicative enzymes
• E.g. norflox
  inhibit DNA gyrase
• ciploflox
  

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• Replication in Eukaryotic cells
• More complex than prokaryotic replication
• Semicoservative ,occurs bidirectional from many
  oigins forming multiple replication bubbles
• Eg- replication of Drosophilia
  chromosomes
• single Ori C ---16 days to
  replicate
• multiple Ori C ---3 min ( 6000
  replication forks )
• Sequence functionally similar to Ori C have been
  identified in yeast are called ARS (
  autonomously replicating sequence )
• ARS span about 300bp ( conserved sequence )
• There are about 400 ARS elements in yeast

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• Eukaryotic DNA polymerases

Type Location Major role
a Nucleus Replication of nuclear DNA Gap filling synthesis of lagging strand
ß Nucleus Proof reading Repair of nuclear DNA
? Mitochondrial Replication of mitochondrial DNA
d Nucleus Replication of nuclear DNA Leading strand synthesis
e Nucleus Repair of nuclear DNA
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• Replication in linear genome
• Problem arise with replication of ends of linear
  genome
• ( Telomers )
• Removal of RNA primer on the lagging strand
  produces a daughter DNA with an incomplete 5
  end
• If not synthesized shorter and shorter daughter
  DNA would result from successive rounds of
  replication
• This problem is solved by the enzyme TELOMERASE

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• Telomers
• Ends of the eukaryotic linear chromosomes
• Contains thousands of hexameric repeats ( TTAGGG
  )
• Some shortening of this telomer is not a problem
  as they donot encode for proteins
• Cell is no longer able to divide is said tobe
  senescent if shortening occurs beyond some
  critical length
• In germ cells ,stem cells as well as in cancer
  cells ,telomers donot shorten the cells do not
  senesce.( due to the presence of Telomerase
  enzyme )

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• Telomerase
• Ribonucleoprotein enzyme ( reverse transcriptase
  ) catalyzing the elongation of the 3 ending
  strand
• Contains a RNA molecule that serves as the
  template for the elongation of the telomeric end
• Highly processive hundreds of nucleotides are
  added before it dissociates

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Thank you