Polymerases

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POLYMERASES

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Polymerases

• DNA-dependent DNA polymerases
• Eg. Prokaryotic and Eukaryotic DNA
  polymerases
• DNA-dependent RNA polymerases
• Eg. RNA polymerases and DNA primase
• RNA-dependent DNA polymerases
• Eg. Reverse transcriptase
• RNA-dependent RNA polymerases
• Eg. RNA replicase

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RNA Replicase

• Reich et al. showed the RNA synthesis even in the
  presence of actinomycin-D (that inhibits the
  DNA-primed RNA synthesis)
• Baltimore and Franklin showed the viral
  production with specific cytoplasmic fractions
• Thus, RNA-primed RNA synthesis was demonstrated
• This enzyme has been named RNA-dependent RNA
  polymerase or RNA Replicase

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All RNA viruses (except retroviruses) encode a
protein since the hosts do not possess it

• Structure of Qß replicase
• Four subunits
• 3 provided by E. coli host (Tu, Ts, and S1)
• 1 in viral RNA genome (MW 65,000)

• Function
• Synthesis in 5 to 3 direction
• Lacks proofreading function
• Requires RNA template
• Specific for RNA of the virus

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• RNA Replicase was isolated by Spiegelman et al.
  from Qß bacteriophage
• This requires RNA template, Mg2, rNTPs
• Qß replicase is tetramer consisting of one Rep
  molecule and three host proteins
• Replicase copies viral () strand to generate a
  (-) strand

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• During synthesis, (-) strand is in contact with
  the () strand only at the polymerization site
• Initiation of several (-) strand occurs before
  the first (-) is complete, and the replicative
  form is branched
• The (-) strands are released and immediately used
  by the replicase to form () strands only

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Reverse Transcriptase
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Extension of the Central dogma
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• Temin and Baltimore (1970) independently
  demonstrated the presence of RT in several avian
  and mouse retroviruses
• RT is a RNA-dependent DNA polymerase
• It is used in rDNA technology to transcribe mRNA
  into dsDNA

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• RT has 3 enzymatic activities
• It copies an RNA molecule to yield dsDNA-RNA,
  using a primer and joining dNTPs in a 3-5
  linkage
• It copies a primed ssDNA to form dsDNA
• It degrades RNA in a DNA-RNA hybrid this is
  called RNase H activity (3 to 5 exoribonuclease
  activity)

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Structure and gene products
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(No Transcript)
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Retrovirus - HIV
(1) Viral genome and reverse
transcriptase enter cell.
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA
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(1) Viral genome and reverse
transcriptase enter cell.
(8) Final viral assembly and budding
take place.
(2) DNA copy synthesized by reverse
transcriptase.
RNA
DNA
(3) RNA degraded second DNA strand
synthesized.
DNA
DNA