dna topoisomerases. dna supercoiling in vivo in most organisms, dna is negatively supercoiled ( ~...

DNA Topoisomerases

DNA Supercoiling in vivo

• In most organisms, DNA is negatively supercoiled (~ -0.06)

• Supercoiling is involved in initiation of transcription, replication, repair & recombination

• Actively regulated by topoisomerases, ubiquitous and essential family of proteins

Chromosomes: the ultimate Gordian knot?

EM by U. Laemmli

Topological issues in DNA replication

Supercoiling and transcription

• In bacteria, gyrase helps maintain negative supercoiling.

• This can help drive transcription in many genes (although gyrase is, itself, downregulated by negative supercoiling).

• Mutations in gyrase are compensated by mutations in topo I to prevent it from removing negative supercoiling.

• Positive supercoils ahead of RNAP, negative supercoils behind?

Bacterial Topoisomerases

VIRAL TOPOISOMERASES: vaccinia (smallpox), phage T4 Topo II

Eukaryotic Topoisomerases

Mechanisms of Type II Topoisomerases

Therapeutic Implications

Gyrase is a good target for antibacterial quinolones (ciproflaxin).

DNA Breakages are toxic…Reversed by tyrosyl-DNA phosphodiesterases (3’ topo Ib

breaks)…How are tdp proteins and other break-repairing proteins

(involved in recombinational repair) involved in resistance to chimiotherapeutic agents?

Topoisomerase II poisons are used in chemotherapy (daunorubicin, doxorubicin, etoposide) as well as Topo I poisons (topotecan)

How to detect topoisomerase activity in a single-molecule assay

is calibrated by measuring the change in DNA extension observed for a unit rotation of the bead

Single turnovers observed at low (10 m) ATP

•Two supercoils relaxed per catalytic turnover•Tcycle displays single-exponential statistics

Processive activity at higher [ATP]

Topo II activity

Magnet rotation applied

•Trelax << Twait single molecule bursts•Processivity on the order of ten cycles

DNA crossovers are the substrate of topo II

Eurkaryotic Topo II does not distinguish (+) and (-) sc

[ATP] and force-dependence of strand passage

Km = 270 M ATPVsat = 3 cycles/sec

•Rate-limiting step coupled to ~1nm motion against the applied force

How do we know this is not torque-related?

Charvin et al., PNAS (2003) 100: 115-120

Decatenation Experiments show similar Kcat

V0 = 2.7 cycles/s, = 1.9 nm

High processivity (commonly 40, up to 80 reported)

Charvin et al., PNAS (2003) 100: 115-120 Enzyme rate is not torque-sensitive

Model: closure of the DNA gap is rate-limiting

Principle of “clamping” experiment

Topo II binds to DNA crossovers

Detection of individual clamping events

(DNA is pre-twisted to the threshold of the buckling transition)

Clamping lifetimes: with Magnesium

Bacterial Topo IV distinguishes (+) and (-) sc

Distributive Processive

Again: is torque driving this effect??

Use braided DNA molecules to measure effect of topology without torque

Charvin et al., PNAS (2003) 100: 115-120

Force-response of bacterial topo IV

L-braids (topologically equivalent to + supercoils) are removed more quickly than R-braids (~ – supercoils)Final R-braid crossover very hard to remove (as opposed to final L-braid crossover.Topo IV cycle less mechanosensitive than topo II cycle.At the same time, characteristic length-scale for work against force at rate-limiting mechanosensitive step involves displacement against force over a distance of ~10 nm (5x greater than topo II)

Charvin et al., PNAS (2003) 100: 115-120

Topo IV can remove R-braids if they supercoil(thus forming L-crossovers)

Charvin et al., PNAS (2003) 100: 115-120

Type I Topoisomerases: a comparison

Topo Ia Topo Ib

Measuring step-size by variance analysis

1. X(t) is the recorded position of the system2. Record many (long) traces and average them together

mean = X = NPvariance = X - X = NP(1-P)2

(t)n

n!___ exp(-t)P(n) =

Random

Observation of RecBCD helicase/nuclease activity

Bianco et al., Nature (2001) 409: 374-378.

Problems with using flow fields a non-linear enzyme rate?

Bianco et al., Nature (2001) 409: 374-378.

UvrD unzips DNA without chewing it up

(conversion assay)

Dessinges et al., PNAS (2004), 101: 6439--6444

At low force DNA hybridization is a problem

Dessinges et al., PNAS (2004), 101: 6439--6444

Unzipping, zipping and hybridization are observed

Dessinges et al., PNAS (2004), 101: 6439--6444

Measuring step-size by variance analysis

mean distance travelled = NPvariance of distance travelled = NP(1-P)2

Like a random walk: N steps with a probability P (small) of moving forward a distance

Repeat the walk a large number of times and average the results together

mean

variance=