Topological Problems in Replication Linear Chromosomes: Telomerase for replication of the ends...
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Transcript of Topological Problems in Replication Linear Chromosomes: Telomerase for replication of the ends...
Topological Problems in Replication
Linear Chromosomes: Telomerase for replication of the ends
Topoisomerases to relieve strain of untwisting and supercoiling
Problem of linear templates
• 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?
3’
5’
5’ 3’
Primer?
5’
5’3’
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
Replication
aaa
aaa
aaa
aa
aa
aaa
+
aaa
aaaa
Telomerase adds more copies of "a’" to 3’ end of strand with overhang
DNA synthesis
a = CCCCAA, a’ = GGGGTT in humans
aa
aaa a’a’a’a’
a’a’a’a’
The segment complementary to the 3’ end of template is not replicated.
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 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.
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 itselfRelaxed 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, L, is partitioned between T and W, i.e.
• L=W+T
• if L = 0, then W= -T
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 = = 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 = -0.05, G=-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
Genes VI : Figure 17-15
One strand passes through a nick in the other strand.
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.
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 fast-
growing bacterial
cells
E.g. every 30 min: Cells divide Replication initiates
Cell cycle in eukarytoes
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.
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
More about cell cycle regulation
• BMB 460: Cell growth and differentiation
• BMB 480: Tumor viruses and oncogenes
• BMB/VSC 497A: Mechanisms of cellular communication