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=