Micromechanical study ofprotein-DNA interactions

and chromosome structure

Assemble and disassemble protein-DNA complexes, observe in real time, control by force

1. DNA looping by E. coli nucleoid protein Fis

2. Micromanipulation of mitotic chromosomes to probe higher-order chromosome structure

(3. Chromatin assembly using Xenopus egg extractsTheoretical model for experiment: Ranjith Padinhateeri)

HMGD (Human)

HU(E. coli)

LEF-1 (Mouse)

IHF (E. coli)

Micromechanical study of protein-DNA interactions

naked DNA

+ sharp bends

Study the action of these proteins via mechanicalresponse of the DNA they are binding to

+ loops

E. coli is filled with DNA

E. coli chromosome = 4.5 Mb = 1.5 mm Nucleoid volume < 1 m3

Concentration of DNA ~ 10 mg/mlDNA is covered with protein

Wang, Possoz, Sherratt Genes Dev 2005 E. coli phase contrast

J Struct Biol 2001, Bar 5 um

E. coli loop domains: classical view

50-500 loops

Each loop10 to 100 kb3 to 30 m

+supercoiling

Loop anchors?

Postow, Hardy, Asuaga, Cozzarelli, Genes Dev 2004

Visualization ofsmall E. coli loop

domains

500 nm

100 nm

Transverse magnetic tweezer 97004 bp 32.8 m dimer of

•built on inverted microscope stage•micropipette holds left 2.8 m bead•right bead under tension applied by magnet off to right•wide range of forces (0.1 to 100 pN)•high position sensitivity (< 10 nm) Jie Yan, Ph.D. 05•Relatively easily combined w/ fluorescence John Graham, Ph.D.

Mag

net

(20

0 m

aw

ay)

FIS (E. coli)20 kD dimerknown for DNA bendingmajor nucleoid protein40,000 dimers/cell during exponential growth 50 M or 1 dimer / 230 bp

Stationary phase~100 dimers/cell Dps dominant

note also HN-S, HU, IHF, MukBEF Reid JohnsonUCLA Medical School

Schneider et al NAR 2001

FIS observed to increase branching of scDNA binds to DNA in clusters

Fis has been suggested to generate “higher-order” DNA folding

0 nM1 M

6 M

13 M

≥1 uM FIS condenses DNA completely below a low critical force

200 nM

Hypothesis: FIS can stabilize DNA loops

Free energy cost vs binding (free) energyF = F0() + f -

Minimum F0 ~ +5 kBT for ~ 80 nm

Loop formation rate exp( – f / kBT )

Looping requires f < 10 kBT / ~ 0.5 pN

FIS+DNA condenses and opens via discrete jumpsFis can stabilize DNA crossings (loops)

(Skoko et al PRL 2005, JMB 2006, Hua Bai)

5 M FIS10 kb DNA

0.3 pN

5 pN

FIS-DNA complexes don’t spontaneously dissociate

Naked50 nM FISBuffer wash

Naked5 uM FISBuffer washThen reduce f

Naked50 nM FIS50 ug/ml DNA250 ug/ml

Naked5 uM FIS50 ug/ml DNAThen reduce f

FIS-DNA complexes even more stable than HU, HMG, NHP6A complexes (see Skoko et al Biochemistry 2004, JMB 2006)

Dissociation of proteinfrom DNA to solution blocked

(hour timescale)

‘Direct transfer’ to a different DNA is the main

disassembly pathway(DNA competes for protein)

Protein-proteincooperativity

Counterioncondensation

http://www.npwrc.usgs.gov/ narcam/idguide/rsnewt.htm

Metaphase

Bar: 20 m

Micromanipulation of individual mitotic chromosomes

stretch

Mitotic chromosomes are elastic “springs”

‘Microspraying’ - kinetic biochemical studiese.g. shift local ionic conditions

(100 mM MgCl2 in culture buffer)

Surprisingly reversible chromosome ‘breathing’ using ionsPoirier et al, J Cellular Biochem 2002

Paulson & Laemmli Cell 12, 817 1977

Stack & AndersonChromosome Res. 9, 175 (2001)

What happens when we cut DNA only?MC nuclease digestion, 0.1 nN initial tension

Cut DNA in mitotic chromosome sufficiently frequently and it falls apart;

classical ‘protein scaffold’ must be disconnected(1 nM AluI AG^CT, PNAS 2002)

0 s 30 90 390270 0 s 60 120 180 250

c

a b

Increasing trypsin digestion Increasing proteinase K digestion

Proteolysis reduces but does not eliminate elastic response

0 s30

90

270

390

100 nM trypsin

0 s

3060

90

120

500 nM proteinase Kd

L.H. Pope, see also experiments of Maniotis 1997, Almagro 2004

Proteolysis leads to a strong swelling of the mitotic chromosome but never breaks or dissolves it

Chromosome still elastic with well-defined shape after >30 min proteolysis

0 s30

60

120 240

480840

1320

Enhanced contrast

1320

Extensive proteinase K digestion

30 nmchromatin fiber

Mitotic Chromosome

linker protein (SMCs ?) approximately 15 kb spacing

Pope: Effects of protease (MBC 2006)Kawamura: Topo II and DNA entanglements (2007)

Chromatin (nucleosome) assembly onto a single 97 kb DNA using Xenopus egg extracts (-ATP) (collaboration with Prof. Rebecca Heald, UC Berkeley)

•Xenopus egg extract solution – assembles chromatin on DNA•32.8 um DNA becomes 3.6 m fiber (400 nucl) in 600 sec•Starting point for further chromatin-based experiments

(Bennink et al, 2001, 2005; Ladoux et al 2000, Wagner et al 2005)

UCLAReid Johnson

NYUDavid RothBernhard Schnurr

UCBRebecca Heald

UICMike Poirier (OSU)Dunja Skoko (NIH)Jie Yan (NUS)Lisa Pope (Cambridge)Hua BaiRyo Kawamura

NULance Min

NSF DMR BIO PHYNIH (via NYU)

Northwestern University