Stuart R.W. Bellamy1, Yana S. Kovacheva1, Ishan Haji Zulkipi1,Guus Harms2, Niels Laurens2, Gijs J.L. Wuite2, Stephen E. Halford1.

The Dynamics of DNA Binding, Looping and Dissociation by the Type II Restriction Endonuclease SfiI

(1) Department of Biochemistry, University of Bristol, UK.

(2) Department of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands.

AbstractAbstract

WT SfiI forms extremely stable loops in the presence of Ca2+. In order to observe

dynamic looping in the presence of divalent metal ion, two catalytically inactive

mutants were generated, D79A and D100A. However, these mutants form stable

loops in the presence of Ca2+. D100A also forms stable loops in the presence of

Mg2+, but D79A forms transient loops under these conditions.

TPM experiments have measured the loop lifetime and loop formation time(s) for the

D79A-DNA complex in Mg2+. Although further data analysis is required, from these

experiments we can start to build up an accurate picture of the kinetics of DNA

looping by SfiI in the presence of Mg2+.

[1] Szczelkun & Halford (1996) EMBO J, 15, 1460-1469

[2] Milsom et al. (2001) JMB, 311, 515-527

[3] Vanamee et al. (2005) EMBO J, 24, 4198-4208

[4] Newman et al. (1998) EMBO J, 17, 5466-5476

[5] van de Broek et al. (2006) NAR, 34, 167-174

[6] Nobbs et. al. (1998) JMB, 281, 419-432

We thank Richard Sessions, Marks Szczelkun and Dillingham and Lucy Catto for ideas and discussion.

ReferencesReferences Acknowledgements

Acknowledgements

SfiI is a Type II restriction endonuclease that acts at the sequence:

GGCCnnnnnGGCCCCGGnnnnnCCGG

+ Mg2+ @ 50oC

Two-site DNA: SfiI acts in cis, looping out the intervening DNA:

SfiI differs from the orthodox Type II enzymes in that it is tetramer in solution and needs to bind two copies of its recognition site before displaying full activity. Consequently, while it can cleave DNA with one SfiI site by acting in trans, it works best on DNA molecules with two sites, by acting in cis. Once bound to two sites, it cuts both before dissociating from the DNA.

One-site DNA: SfiI acts in trans, bridging two DNA molecules:

IntroductionIntroduction

SfiI is a prototype system for studying DNA looping. However, dynamic looping has only been detected in EDTA (t1/2 = 4 mins [1]). In the presence of Ca2+, SfiI forms very stable loops (t1/2 = >7 hours [2]). The association/dissociation kinetics of SfiI were examined here by three methods:

1) Gel shift; 2) FRET; 3) Tethered particle motion (TPM)

In Ca2+, DNA in the SfiI-DNA complex is not displaced by competitor

DNA, even after 24 hours

The dissociation of DNA from wild-type (WT) SfiI was followed in the presence of Ca2+ to prevent cleavage. WT SfiI (5 nM) was incubated with HEX-labelled 35-mer (10 nM), then a 10X excess of unlabelled 21-mer (100 nM) was added. The complexes containing two labelled 35-mers, one labelled 35-mer and one unlabelled 21-mer, and the free labelled 35-mer were then resolved on a polyacrylamide gel.

0 1 2 4 6 24

Time (hours)

*

***

1) Gel-shift: Dissociation of DNA from Sfi-DNA Complex1) Gel-shift: Dissociation of DNA from Sfi-DNA Complex

Substrate: 21-mer & 35-mer: HEX-TCGATCCATGTGGCCAACAAGGCCTATTTGTCGAT AGCTAGGTACACCGGTTGTTCCGGATAAACAGCTA

**

*

10-foldIncubate for 1-24 hrs

Incubate for 30 min

*+

*+

*

Rates of loop formation and dissociation can only be measured under dynamic equilibrium conditions a new approach is needed.

Can use catalytically inactive mutants of SfiI that can bind DNA but cannot cleave it. This could also allow binding reactions to be performed in the presence of Mg2+.

The crystal structure of SfiI bound to DNA in Ca2+ [3] has only one metal ion in its active site, coordinated by D79 and D100, but not in position for catalysis. In contrast, BglI [4] has two metal ions, ideally placed for hydrolysis. In the expectation that D79 and D100 bind the metal ions required for catalysis by SfiI, both residues were mutated to A to give D79A and D100A mutants.

TT G

Asp79Asp100

Lys102Ca12+

Glu55

SfiI Catalytically Inactive MutantsSfiI Catalytically Inactive Mutants

Lys144

T

A A

Asp116Asp142

Glu87

Ca22+

Ca12+

Lys144

SfiI @ GGCCTTGTTGGCC (x2) BglI @ GCCTAATAGGC (x1)

Pictures by Dr Richard Sessions

Time (minutes)0 0.5 1

[DN

A] (

nM)

0

1

2

3

4

5

10 20 30

WT D79A D100A

Cut 2 sites

Cut 1 site

Activities of D79A & D100A were tested by adding 5 nM enzyme to 5 nM DNA (2 SfiI sites) in Mg2+-buffer at 50 ºC. No cleavage was observed with either mutant, even after overnight incubation.

Activity and Binding of D79A and D100A MutantsActivity and Binding of D79A and D100A Mutants

Time (minutes)0 100 200 300

[DN

A] (

nM)

0

1

2

3

4

5

1400Time (minutes)

0 100 200 300

0

1

2

3

4

5

1400

Substrate

35-mer

D100AWT

21-mer

DNA binding was analysed by gel shift. WT SfiI or D100A (D79A not shown) were added to the 21-mer and 35-mer in varied ratios, from 100% 21-mer to 100% 35-mer. Across this range, three DNA-protein complexes were observed: with two 21-mers; one 21-and one 35-mer; two 35-mers. In addition, AUC data (not shown) shows that both mutants are tetramers. Hence, like the WT, D79A and D100A are tetramers that bind two cognate sites.

Substrate Substrate

Time (hours)

*

***

Dissociation in Ca2+ - D100ADissociation in Ca2+ - D100A

Double exponential (amplitudes in brackets): k1 = 8.1 h-1 (33%); k2 = 0.92 h-1 (67%)

A

B

C

0 0.3 0.6 1 2 3

Time (hours)

[La

be

lled

DN

A]

(nM

)

A

B

C

0 0.5 1 1.5 2 2.5 30

2

4

6

8

10

Gel shifts were performed, as with WT SfiI, to follow the dissociation of DNA from D100A. Unlike the WT, dissociation of the labelled DNA was observed. The gels were analysed with respect to the labelled DNA using ImageQuant, and the decrease in the concentration of complex with two labelled 35-mers was fitted to both a single (red) and double (blue) exponential.

Reduced 2 values for single and double exponential are 0.1738 and 8.137 x 10-5

double exponential is a much better fit

Single exponential: k = 3 h-1

Dissociation in Ca2+ - ModellingDissociation in Ca2+ - Modelling

The reactions of the pathway were modelled in Berkeley Madonna by numerical integration. This revealed the decrease in EA2 is a single exponential process. Hence, this scheme doesn’t account for the dissociation kinetics observed above.

A scheme was derived for the binding reactions of SfiI. Labelled DNA (A) may dissociate from the EA2 complex to give first EA and then free E. However, the addition of excess unlabelled DNA (B) can generate first a complex with one labelled and one unlabelled DNA (EAB) and then one with two unlabelled DNA molecules (EB2).

Slow conversion of an alternative conformation of the SfiI-DNA complex, E*A2, to EA2, results in slow dissociation of DNA

If an extra step is included, the formation of E*A2 by a conformational change in EA2, the decrease in EA2 is double exponential when k-x<k-a. Thus this extra step is required to account for the dissociation of DNA from D100A. For WT SfiI, k-x<<k-a, hence very little dissociation is observed.

k-b2.kb.A

EA2 EA

E EB

EABka.A

2.k-a ka.B

k-a

ka.B

2.k-a

2.kb.B

k-b

k-aka.A

EB2

Pathway 1

Pathway 2k-b2.kb.A

EA2 EA

E EB

EABka.A

2.k-a ka.B

k-a

ka.B

2.k-a

2.kb.B

k-b

k-aka.A

kx k-x

E*A2

EB2

For D100A and D79A, E:DNA complexes were formed by mixing 25 nM enzyme and 50 nM Alexa350 21-mer. Then a 10X excess of unlabelled 21-mer was added. The decrease in FRET was fitted to either a single (red) or double (blue) exponential.

Time (hours)

0 0.5 1 1.5 2 2.5 3

Re

lativ

e fl

uore

sce

nce

0.8

0.85

0.9

0.95

1

Time (hours)

0 0.5 1 1.5 2 2.5 3

Re

lativ

e fl

uore

sce

nce

0.85

0.9

0.95

1

FRET: Dissociation in Ca2+FRET: Dissociation in Ca2+

k1 = 6.7 h-1 (43%) k2 = 0.57 h-1

(57%)k1 = 28 h-1 (23%)

k2 = 1.4 h-1 (77%)

D79AD100A

Trp250

Trp8585 Å

30 ÅDissociation of DNA from the SfiI-DNA complex was also followed by FRET. The intrinsic Trp fluorescence of SfiI (3 Trps/subunit) was exploited for DNA-protein FRET, using a 21 bp oligo labelled at its 5’ end with Alexa350. On exciting the Trps at 290 nm, an increase in the emission from the Alexa350 21-mer was observed. Addition of excess unlabelled oligo then resulted in a decrease in FRET.

2) DNA – Protein FRET2) DNA – Protein FRET

Trp232

FRET: Association and Dissociation in Mg2+FRET: Association and Dissociation in Mg2+

FRET was also used to follow association and dissociation in Mg2+-buffer. For the association reactions, E (12.5 – 250 nM D79A or D100A) was titrated against fixed [DNA] (20 nM Alexa350 21-mer) in the stopped-flow. Dissociation reactions were performed as above.

D79A

D100A

k1 = 15 h-1 (28%)k2 = 0.91 h-1 (72%)

k1 = 0.2 s-1 (85%) k2 = 0.04 s-1 (15%)

In Mg2+, DNA binds more weakly to D79A than to D100A and dissociates much faster (50-fold) from D79A than from D100A.

BINDING DISSOCIATION

250 nM125 nM50 nM

12.5 nM

Time (seconds)

0 0.1 0.2 0.3

Flu

ores

cenc

e U

nits

24

26

28

30

32

34

250 nM125 nM50 nM

12.5 nM

Time (seconds)

0 0.1 0.2 0.3

Flu

ore

sce

nce

Uni

ts

22

24

26

28

30

32

Time (hours)0 0.5 1 1.5 2 2.5 3

Rel

ativ

e flu

ores

cenc

e

0.75

0.8

0.85

0.9

0.95

1

Time (seconds)0 20 40 60 200 400 600

Rel

ativ

e flu

ores

cenc

e

0.85

0.9

0.95

1

TPM tracks the Brownian motion of a bead tethered by a single DNA molecule by video microscopy. Changes in DNA length caused by looping causes a change in Brownian motion, which is monitored by measuring the RMS motion of the bead. The motion of unlooped tethers is larger than the looped.For example, data from another looping enzyme, NaeI [5]:

3) Tethered Particle Motion (TPM)3) Tethered Particle Motion (TPM)

DIGBIO

~ 250 bp ~ 250 bp~ 500 bp

SfiI1 SfiI2

Glass surfaceANTI-DIGDIG

BIOTIN

Streptavidin-coated bead

Unlooped Looped

RMS motion (nm):

The lifetime of the loop and the loop formation time can be determined from the time spent in the looped and unlooped states. In addition, the association rate of E and DNA can be measured at low [E] from the time taken to form a loop after adding the enzyme.

A DNA substrate was designed for SfiI with biotin at one end (to attach to the bead) and digoxygenin (DIG) at the other (to attach to the glass surface):

Looped

Unlooped

SfiI flown into the flow chamber

SfiI forms a loop in the DNA

0 10 20 30 40 500

50

100

150

200

250

300

RM

S (

nm)

t (min)

0.5 sec filtered data trace Binary trace

# counts

TPM experiments were performed in Ca2+ for WT SfiI, D79A & D100A. For all three enzymes, loops were formed but not released, even after 40 mins (not shown). This is consistent with the gel-shift and fluorescent data.

WT SfiI, D79A & D100A in Ca2+WT SfiI, D79A & D100A in Ca2+

With D79A in Mg2+, transitions between the two distinct RMS states occur over the minute time-scale. Hence under these conditions dynamic looping is observed. This agrees with the solution kinetics, which showed that, in Mg2+, D79A binds DNA weakly and dissociates from it rapidly.

D79A in Mg2+D79A in Mg2+

0.01 nM D100A in Mg2+: kon ~ 108 M-1s-1

Unlooped

Looped

In Mg2+, D100A again formed loops that never dissociated, also matching the solution kinetics. At low [D100A], the association rate (kon) of E and DNA was measured and found to be near the diffusion controlled limit.

D100A in Mg2+D100A in Mg2+

D79A in Mg2+ - Loop Formation and Duration D79A in Mg2+ - Loop Formation and Duration

The loop formation time (form) is a double exponential process:

[D79A] (nM) off form1 form2

0.033 16.90 ± 0.04 99 ± 2 1.81 ± 0.01

0.1 20.4 ± 0.1 73 ± 2 1.61 ± 0.02

0.33 14.83 ± 0.04 36 ± 2 1.94 ± 0.02

Analysis of measured loop duration times reveals that loop dissociation is a single exponential process, giving a lifetime of the D79A-DNA looped complex (off) of 20 s. This corresponds to a loop dissociation rate (koff) of 0.05 s-1, similar to other Type II enzymes [5].

Long time constant form1 - dependent on [E]

SfiI in free solution binding to either of the two sites on the DNA dependent on the diffusion of enzyme to the DNA molecule

Short time constant form2 - independent of [E]

SfiI already bound at one site capturing the second site in cis dependent on the segmental diffusion of the DNA

DNA release Unlooped DNA

Looped DNA

WT SfiI in Mg2+WT SfiI in Mg2+

TPM can also be used to study the turnover of DNA by WT SfiI in Mg2+-buffer at 25 ºC. The release of the bead is monitored, which occurs when SfiI has cut the DNA and released the ends. Hence, this assay should measure product release rather than hydrolysis.

Fraction of non-cleaved tethers vs time:

The decrease in the fraction of non-cleaved tethers over time is a single exponential, with a time constant of ~50 min, giving a bead release rate of 0.02 min-1. From previous kinetic studies on DNA cleavage by SfiI at 25 ºC [6], the rate constant for the hydrolytic step is ~30 min -1, very much faster than the bead release rate, while that for subsequent dissociation of the cleaved product is ~0.01 min-1, in good agreement with the bead release rate.

TPM is a fair representation of solution kinetics