Enzymology of Retroviral Replication

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• Enzymology of Retroviral Replication
• RT Polymerization
• RT Inhibition
• RT Strand Transfer
• IN Processing
• IN Integration

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ssRNA
ds-DNA
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Polymerase Mechanism

• Processivity continued turnover without
  dissociation from DNA
• 2 different kinds of steady-state turnover
• Continuous (Processive)
• Dissociative
• Processivity measures average number of
  continuous turnovers per initiation (binding)
  event

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Single-Turnover

• Difficult to quantify processive turnovers gels
  give long, messy smears
• How does enzyme work at EACH turnover?
• Therefore must design experiments to STOP or
  pause after a single pass through

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2 Requirements

• Property of Substrate
• Oligonucleotides w/ defined sequence
• Single nucleotide incorporation
• Property of Enzyme (cant change this)
• Rate limiting dissociation

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Burst Kinetics
S
S

• E ES EP E ES
• First turnover fast, then slow steady-state
• Therefore burst of products at start of
  reaction defines a single-catalytic event

P
slow
fast
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44 nM RT 200 nM DNA
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/- DNA Trap - kpol 15 s-1 - amplitude
decrease by 10.7 - ampkpol/(kpolkoff) -
koff2 s-1
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ddATP /- dCTP (next nucleotide) - no dCTP
kss .18 s-1 - w/ dCTP kss .019 s-1
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• Induced Fit
• Distinct conformations for
• With no dNTP bound
• With ANY dNTP bound
• With correct dNTP bound
• Only last is kinetically competent

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• Low Fidelity
• Lacks Proof-reading exonuclease
• Advantages of hypermutability
• Small genome size
• Guarantees a single mismatch per virus

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• Simultaneous RNase-H activity

25-26
45
38-39
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Burst Rates unchanged Burst Amplitude reduced
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dATP bound TIGHTER with Cl-TIBO w/ slow
incorporation rate
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Processing
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GTCA
AC
Processing
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GTCA
OH
HO
AC
Joining
Processing
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Disintegration
Target
HO
AC...
U3
AA
TG...
Viral
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1.0
0.5 µM DNA, 0.4 M NaCl
0.25 µM DNA, 0.25 M NaCl
Amplitude
0.5
0
IntegraseDNA
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Photo detector
Laser
Skippy
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(-) DNA
() DNA
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(-) DNA
() DNA
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Apparent M.W. (kDa)
Apparent M.W. (kDa)
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15
Percent Total Mass
10
5
20
60
100
140
180
20
60
100
140
180
Molecular Volume (nm3)
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