Topological Problems in Replication
Linear Chromosomes: Telomerase for
replication of the ends
Topoisomerases to relieve strain of
untwisting and supercoiling
Problem of linear templates
3’
5’
5’
Replication
3’
5’
3’
5’
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?
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
Telomerase adds repeats back to
replicated telomeres
aaa
aaa
Replication
aaa
aa
+
aa
Telomerase adds more
copies of "a’" to 3’ end of
strand with overhang
aaa
aa
The segment complementary to the
3’ end of template is not replicated.
a = CCCCAA,
a’ = GGGGTT
in humans
aaa
a’a’a’a’
DNA synthesis
aaaa
aaa
a’a’a’a’
Replicated telomeres are primers for
telomerase
Telomerase adds 1 nt at a time, using an
internal RNA template
Telomeric repeats form a primer for
synthesis of the complementary strand
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
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.
Different topological forms of DNA
Genes VI : Figure 5-9
Negative and positive supercoils
• Negative supercoils twist the DNA about its axis in the
opposite direction from the clockwise turns of the righthanded (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.
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)
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 W=0
Negative supercoils, W is negative
Positive supercoils, W is positive
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
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.
•
• if
DL=DW+DT
DL = 0, then DW= -DT
Relationship between supercoiling and twisting
Figure from M. Gellert; Kornberg and Baker
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)
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
unwinding
Thus negative supercoiling could favor
initiation of transcription and initiation of
replication.
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.
Topoisomerase I: nicking & closing
One strand passes through a nick in the other strand.
Genes VI : Figure 17-15
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 reseal the break.
TopoII: double strand break and passage
When should a cell start replication?
Bacteria: Rate of cell doubling
determines frequency of initiation
Eukaryotes: Cell cycle control
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
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 < 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.
Multiple
replication
forks in
fastgrowing
bacterial
cells
E.g. every 30 min:
Cells divide
Replication initiates
Cell cycle in eukarytoes
S phase
DNA synthesis
Preparation
for replication
6-8 hrs
~12 hrs
G0
G1
G2
3-4 hrs
quiescent
cells
2nDNA
4nDNA
<1hr
M=mitosis
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 nonexpressed genes replicate late.
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 multisubunit, cyclin-dependent protein kinase.
• Mechanism:
– Increased amount of cyclin
– Correct state of phosphorylation of the kinase
More about cell cycle regulation
• BMB 460: Cell growth and differentiation
• BMB 480: Tumor viruses and oncogenes
• BMB/VSC 497A: Mechanisms of cellular
communication