Topological Problems in Replication

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Topological Problems in Replication

• Linear Chromosomes Telomerase for replication of
  the ends
• Topoisomerases to relieve strain of untwisting
  and supercoiling

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Problem of linear templates
Replication
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Primer?

• Since a primer is required, how do you initiate
  replication at the 5 terminus of a DNA chain?
• How do you prevent progressive loss of DNA from
  the ends after replication?

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Solutions to the problem of linear templates

• Convert linear to circular DNA
• Attach a protein to 5 end to serve as primer
• Make the ends repetitive, e.g. telomeres, and add
  more DNA after replication

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Telomerase adds repeats back to replicated
telomeres
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Replication
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Telomerase adds more copies of "a" to 3 end of
strand with overhang
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The segment complementary to the 3 end of
template is not replicated.
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DNA synthesis
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a CCCCAA, a GGGGTT in humans
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Replicated telomeres are primers for telomerase
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Telomerase adds 1 nt at a time, using an internal
RNA template
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Telomeric repeats form a primer for synthesis of
the complementary strand
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Topoisomerases

• Topoisomerase I relaxes DNA
• Transient break in one strand of duplex DNA
• E. coli nicking-closing enzyme
• Calf thymus Topo I
• Topoisomerase II introduces negative
  superhelical turns
• Breaks both strands of the DNA and passes another
  part of the duplex DNA through the break then
  reseals the break.
• Uses energy of ATP hydrolysis
• E. coli gyrase

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Supercoiling of topologically constrained DNA

• Topologically closed DNA can be circular
  (covalently closed circles) or loops that are
  constrained at the base
• The coiling (or wrapping) of duplex DNA around
  its own axis is called supercoiling.

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Different topological forms of DNA
Genes VI Figure 5-9
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Negative and positive supercoils

• Negative supercoils twist the DNA about its axis
  in the opposite direction from the clockwise
  turns of the right-handed (R-H) double helix.
• Underwound (favors unwinding of duplex).
• Has right-handed supercoil turns.
• Positive supercoils twist the DNA in the same
  direction as the turns of the R-H double helix.
• Overwound (helix is wound more tightly).
• Has left-handed supercoil turns.

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Components of DNA Topology Twist

• The clockwise turns of R-H double helix generate
  a positive Twist (T).
• The counterclockwise turns of L-H helix (Z form)
  generate a negative T.
• T Twisting Number
• B form DNA ( bp/10 bp per twist)
• A form NA ( bp/11 bp per twist)
• Z DNA - ( bp/12 bp per twist)

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Components of DNA Topology Writhe

• W Writhing Number
• Refers to the turning of the axis of the DNA
  duplex in space
• Number of times the duplex DNA crosses over
  itself
• Relaxed molecule W0
• Negative supercoils, W is negative
• Positive supercoils, W is positive

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Components of DNA Topology Linking number

• L Linking Number total number of times one
  strand of the double helix (of a closed molecule)
  encircles (or links) the other.
• L W T

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L cannot change unless one or both strands are
broken and reformed

• A change in the linking number, DL, is
  partitioned between T and W, i.e.
• DLDWDT
• if DL 0, then DW -DT

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Relationship between supercoiling and twisting
Figure from M. Gellert Kornberg and Baker
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DNA in most cells is negatively supercoiled

• The superhelical density is simply the number of
  superhelical (S.H.) turns per turn (or twist) of
  double helix.
• Superhelical density s W/T -0.05 for
  natural bacterial DNA
• i.e., in bacterial DNA, there is 1 negative S.H.
  turn per 200 bp
• (calculated from 1 negative S.H. turn per 20
  twists 1 negative S.H. turn per 200 bp)

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Negatively supercoiled DNA favors unwinding

• Negative supercoiled DNA has energy stored that
  favors unwinding, or a transition from B-form to
  Z DNA.
• For s -0.05, DG-9 Kcal/mole favoring
  unwindingThus negative supercoiling could favor
  initiation of transcription and initiation of
  replication.

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Topoisomerase I

• Topoisomerases catalyze a change in the Linking
  Number of DNA
• Topo I nicking-closing enzyme, can relax
  positive or negative supercoiled DNA
• Makes a transient break in 1 strand
• E. coli Topo I specifically relaxes negatively
  supercoiled DNA. Calf thymus Topo I works on
  both negatively and positively supercoiled DNA.

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Topoisomerase I nicking closing
One strand passes through a nick in the other
strand.
Genes VI Figure 17-15
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Topoisomerase II

• Topo II gyrase
• Uses the energy of ATP hydrolysis to introduce
  negative supercoils
• Its mechanism of action is to make a transient
  double strand break, pass a duplex DNA through
  the break, and then re-seal the break.

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TopoII double strand break and passage
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When should a cell start replication?

• Bacteria Rate of cell doubling determines
  frequency of initiation
• Eukaryotes Cell cycle control

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Control of replication in bacteria

• Bacteria re-initiate replication more frequently
  when grown in rich media.
• Doubling time of a bacterial culture can range
  from 18 min (rich media) to 180 min (poor media).
• Time required for replication cycle is constant.
• C period
• time to replicate the chromosome 40 min
• D period
• time between completion of DNA replication and
  cell division 20 min
• C D 1 hour

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Multiple replication forks allow shorter doubling
time

• Doubling time for a culture can vary, but time
  for replication cycle is constant!
• Variation is accomplished by changing the number
  of replication forks per cell.
• If doubling time of culture is lt 60 min, then a
  new cycle of replication must initiate before the
  previous cycle is completed.
• Initiate replication at same frequency as cell
  doubling, e.g. every 30 min.

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Multiple replication forks in fast-growing
bacterial cells
E.g. every 30 min Cells divide Replication
initiates
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Cell cycle in eukarytoes
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Multiple replicons per chromosome

• Many replicons per chromosome, with many origins
• Replicons initiate at different times of S phase.
  
• Replicons containing actively transcribed genes
  replicate early, those with non-expressed genes
  replicate late.

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Regulation at check-points

• Critical check-points in the cell cycle are
• G1 to S
• G2 to M
• Passage is regulated by environmental signals
  acting on protein kinases
• e.g., if enough dNTPs, etc for synthesis are
  available, then a signal activates a
  multi-subunit, cyclin-dependent protein kinase.
• Mechanism
• Increased amount of cyclin
• Correct state of phosphorylation of the kinase

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More about cell cycle regulation

• BMB 460 Cell growth and differentiation
• BMB 480 Tumor viruses and oncogenes
• BMB/VSC 497A Mechanisms of cellular
  communication