Rusling, D.A., Pernstich, C., Catto, L.E., #Laurens, N., #Wuite, G.J.L., and Halford, S.E.

1The DNA-Protein Interactions Unit, Department of Biochemistry, University of Bristol, Bristol, BS8 1TD UK.

#Department of Physics and Astronomy, VU University, Amsterdam, 1081 HV, The Netherlands.

Exploring DNA-looping dynamics by the FokI restriction endonuclease

The FokI restriction endonucleaseFokI is a Type IIS restriction enzyme that binds to an asymmetric DNA sequence and cleaves 9 and 13 bases downstream of this site on the top and bottom strands, respectively (Fig. 1). The protein is composed of two domains: an N-terminal DNA-binding domain and a C-terminal catalytic domain (Fig. 2). The latter contains the functions to cut one strand of DNA and must swing out and dimerise with the catalytic domain of another FokI monomer to cut both strands. This dimerisation occurs once the enzyme is bound to its correct DNA sequence and its partner can be either a free monomer from solution or more favourably, another DNA-bound monomer, trapping the DNA in a loop. Consequently FokI cuts DNA with two sites faster than DNA with one.

Fig. 1: FokI recognition and cleavage site

5’-GGATGNNNNNNNNNNNNNN3’-CCTACNNNNNNNNNNNNNN

Catalytic domain DNA-binding domain

Fig. 2: FokI crystal structure1.

But how does FokI bring two DNA sites together?

parallel antiparallel

Fig. 3: Model for FokI dimerisation in trans with sites in either a parallel or antiparallel orientation2

It has been suggested that in trans, on separate DNA molecules, FokI prefers to generate a synaptic complex where the two DNA recognition sites are held in a parallel and not antiparallel alignment2 (Fig. 3).

However, it has yet to be elucidated which arrangement FokI prefers to generate with the two sites in cis, on the same DNA molecule. By examining the activity of FokI on plasmids that contain sites in either an inverted or directly repeated orientation (Fig. 4) it was possible to determine whether FokI prefers to loop two-site DNA with sites in a parallel or antiparallel alignment.

Experimental strategy

apex

Inverted FokI sites; IF181-190 Directly repeated FokI sites; DF181-190

apex

GGATGNNNNNNNNNNNNN NNNNNNNNNNNNNCATCC

CCTACNNNNNNNNNNNNN NNNNNNNNNNNNNGTAGG

Generates a parallel arrangement

GGATGNNNNNNNNNNNNN NNNNNNNNNNNNNGGATGNNNNNNNNNNNNNN

CCTACNNNNNNNNNNNNN NNNNNNNNNNNNNCCTAGNNNNNNNNNNNNNN

Generates an antiparallel arrangement

Supercoiled plasmids were designed with relatively short intersite spacings (<200bp) so as to restrict the juxtaposition of the sites to the apex of the plasmid superhelix. In this way two sites in an inverted orientation will only align the two sites in a parallel arrangement, while sites in a directly repeated orientation will only align in an antiparallel arrangement (Fig. 4). Since a classic signature of DNA looping is a cyclic dependence on intersite spacing each arrangement was investigated with site spacings of either 181, 185 or 190 bp apart (about one helical turn). The activity of FokI on all six plasmids was examined.

181/185/190 bp

Fig. 4: Supercoiled substrates used to determine the preferred arrangement of binding sites

181/185/190 bp

FokI reactions under steady-state conditions ([DNA]>[E])It was seen that the amount of open circle (OC) DNA released from the enzyme during each reaction varied cyclically with intersite spacing (Fig. 5; right graph). This provides an indication of the instability of the enzyme-DNA complex since an unstable complex will dissociate from the DNA before both strands have been cut. The data suggests that FokI is looping the DNA in both arrangements, however the antiparallel complexes generate more OC DNA and are likely to be less stable.In contrast, the velocity of the reaction on each plasmid failed to show any cyclic dependence, probably because under steady state conditions the rate-limiting step of the reaction is the release of cleaved product (Fig. 5; left graph).

180 182 184 186 188 190

Lib

era

ted

OC

DN

A

0

1

2

3

180 182 184 186 188 190

Rat

e o

f cle

ava

ge (

mol

/mo

l/min

)

0

0.2

0.4

0.6

0.8

1

1.2

Fig. 5: Steady-state experiments. Reactions were initiated by mixing 2 nM FokI with 10 nM DNA in reaction buffer

Site spacing

parallelanti-parallel

FokI reactions under single-turnover conditions ([E]>>[DNA])

0 20 40 60 80

kobs

(s-

1)

0

2

4

6

Enzyme (nM)

0 20 40 60 80

0

2

4

0 20 40 60 80

0

2

4

6

The exponential rate constants from these reactions were found to increase with [FokI] in a sigmoidal fashion and the data were fitted to the Hill equation (Fig. 6). In each case the best fit for each plasmid was obtained with a Hill coefficient close to 2 (i.e. one monomer per site). For all plasmids a KD for the binding of FokI to an individual site was found to be 4 nM. In contrast, the maximal cleavage rate (kmax) was faster for the plasmids containing sites in an antiparallel (red data points; ~6 s-1) compared to parallel arrangement (black data points; ~3 s-1). This maximal cleavage rate incorporates at least two steps: the protein-protein association to trap the DNA loop (kloop) followed by phosphodiester bond hydrolysis (kcleave) (Fig. 9). Which of these limts Kmax to either 3 or 6 s-1 has yet to be established.

Fig. 6: Post-mix experiments. Reactions were started by mixing 5-75 nM FokI with 2.5 nM DNA in reaction buffer.

parallelanti-parallel

181 bp 185 bp 190 bp

time (sec)

0 0.4 0.8 1.2 1.6 2

DN

A (

nM)

0

0.4

0.8

1.2

1.6

2pIF185pDF185fast rate

slow rate

By adding Mg2+ to a solution containing “pre-mixed” FokI and DNA it was possible to bypass the looping step. For all of the plasmids a small fraction of the DNA was cleaved at an extremely fast rate (20 s-1; Fig. 7). As this must be due to a complex in which the enzyme is already bound to the DNA it can be assigned to the phosphodiester hydrolysis step (kcleave). The maximal rate constants from post-mix reactions (kmax=3 or 6 s-1)

can therefore be assigned to kloop for loop closure. FokI therefore captures loops with sites in an antiparallel arrangement faster than in parallel (Fig. 9).

Fig. 7: Pre-mix experiments. 125 nM FokI and 2.5 nM DNA were incubated for 20 minutes before starting the reaction with 10 mm Mg2+

By-passing the looping step

20 s-13 s-1

Inverted FokI sites; IFs

6 s-1 20 s-1

Direct repeat FokI sites; DFs

E2S L Pkloop

k-loop

kcleave

kmax

Fig. 8: Looping and cleavage steps by FokI Fig. 9: FokI on DNA with two sites in different orientations

Model for looping in either arrangement

parallel antiparallel

Tethered particle motion (TPM)

Fig. 10: Setup of TPM and tracking pattern (insert)

unloopedlooped

unlooped?looped?

In this technique beads are attached to one end of a single DNA molecule, whilst the other end is tethered to a glass surface. Such beads undergo Brownian motion that is restricted by the reach of the DNA tether. Changes in the tether length (i.e. by looping of the DNA) are observed as a change in Brownian motion. The motion of the beads are tracked using standard brightfield microscopy (Fig. 10).

Fig. 11: RMS motion showing looped and unlooped states

+ Ca2+

Typical traces for DNA tethers containing either inverted or directly repeated FokI sites are shown in Fig. 11. Two defined states are observed for the tether containing inverted sites, with an expected change in RMS. In contrast, the data is not well defined for the tether containing directly repeated sites, suggesting that the dynamics of the complexes are too fast too measure (< 0.68 sec). The loop lifetime of FokI complexes with sites in an antiparallel arrangement is therefore shorter.

Example IF190 tether

Example DF190 tether

FokI forms looped complexes on DNA with two sites in either an inverted or directly repeated orientation. However the complexes in a directly repeated orientation, which generate an antiparallel arrangement of sites, were less stable (Fig. 5).

Under single-turnover conditions, the maximal rate of the reaction started by adding enzyme to DNA was faster when the sites were in an antiparallel arrangement (6 s-1) compared to a parallel arrangement (3 s-1) (Fig. 6).

For all substrates regardless of site orientation, reactions initiated by adding Mg2+ to pre-mixed DNA/enzyme resulted in very rapid cleavage of some of the DNA (~20 s-1) which must correspond to phosphodiester bond hydrolysis (Fig. 7).

A scheme that accounts for these two processes has been proposed (Fig. 9) and suggests that FokI captures loops with sites in an antiparallel arrangement faster than in parallel. However as the antiparallel complexes are less stable (Fig. 5) then loop breakage must also be faster for these complexes.

Tethered particle motion experiments confirm the shorter lifetimes of the complexes generated in an antiparallel arrangement (Fig. 11).

1. Wah et al. (1997) Nature, 388:97-100. 2. Vanamee et al. (2001) J. Mol. Biol. 309:69-78

Conclusions

References