DNA Topoisomerases
DNA Supercoiling in vivo
• In most organisms, DNA is negatively
supercoiled (s ~ -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 mm) ATP
•Two supercoils relaxed per catalytic turnover
•Tcycle displays single-exponential statistics
Processive activity at higher [ATP]
Magnet rotation applied
Topo II activity
•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 mM ATP
Vsat = 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
High processivity (commonly 40, up to 80 reported)
V0 = 2.7 cycles/s, D = 1.9 nm
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
Random
(lt)
___n exp(-lt)
P(n) =
n!
1. X(t) is the recorded position of the system
2. Record many (long) traces and average them together
mean
=
<X>
= NPD
variance = < (X - < X >)2 > = NP(1-P)D2
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
Like a random walk: N steps with a probability P (small) of moving forward a distance D
Repeat the walk a large number of times and average the results together
mean distance travelled
= NPD
variance of distance travelled = NP(1-P)D2
variance
mean
=D