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