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’ 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 Telomerase adds more copies of "a’" to 3’ end of strand with overhang aa + aa aaa aa The segment complementary to the 3’ end of template is not replicated. aaa a’a’a’a’ DNA synthesis aaaa a = CCCCAA, a’ = GGGGTT in humans 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 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 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. DL=DW+DT if 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 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