Science - USA (2022-06-03)

side of its HEAT-repeat solenoid (fig. S15A).
Homologous DNA interactions are also con-
served for cohesin ( 15 – 17 ). We confirmed the
importance of these DNA interactions for
in vivo condensin function (fig. S15B and table

S2), DNA-dependent ATPase stimulation

(fig. S15C), and DNA loop extrusion (fig. S15D).

Although the nucleotide-free apo structure of

condensin adapts a markedly different confor-

mation, most of the local surface of Ycs4 that

contacts the DNA backbone remains accessi-

ble and unchanged in the absence of nucleo-

tide (fig. S15A), which supports the conclusion

that kleisin chamber I entraps DNA also in the

ATP-free state.

Shaltielet al., Science 376 , 1087–1094 (2022) 3 June 2022 4of8

Fig. 3. Cryo-EM structure of ATP-engaged con-

densin with DNA bound in both kleisin chambers.

(A) Density map of the DNA-bound condensin core

module composed of Smc2head(blue), Smc4head

(red), Ycs4 (yellow), and Brn1 (green) resolved to a

nominal resolution of 3.5 Å. (B) Density maps of

the DNA-bound condensin peripheral module com-

posed of Ycg1 (orange) and Brn1 (green) resolved to

a nominal resolution of 3.9 Å. (C) Path of the Brn1

kleisin subunit through the condensin holo complex.

Of the 754ScBrn1 residues, 376 can be built into

the model; unresolved connections are indicated

as dotted lines. (D) Structural comparison of the

core module in the nucleotide-free apo (PDB ID:

6YVU) and ATP-bound state (PDB ID: 7QEN). The

DNA double helix has been docked into chamber I in

the apo state. (E) Schematic representation of

the tilting motion that feeds DNA held in kleisin

chamber I into the coiled-coil lumen upon ATP

binding. (F) Agarose gel electrophoresis mobility shift

of SDS-resistant condensin–DNA catenanes after

bBBr cross-linking the Smc2–Smc4–Ycs4 lumen in

the absence or presence of nucleotide. AMP-PNP,

adenylyl-imidodiphosphate.

2 ATP

ATPTP

DNA (EtBr)

AB

CD

E

F

Apo core +ATP core

Core

DNA

Brn1

Ycs4

Smc4

Smc2

Ycg1

Periphery

Brn1 DNA

Ycg1

65 aa

110 aa

51 aa

Chamber I

Chamber IA

Safety belt

Chamber II

Brn1Ycs4

55 aa

Brn1C

2 ATP

DNA docked into chamber I

AMP-PNP

ATP

–+++bBBr

+

+

Brn1

N Brn1

Ycs4

Brn

(^1) Ycg1
ATP
K639C
Smc2
N985C
E356C
V721C
Smc4
Ycs4
S238C K1170C
10
8
kbp 6
Fig. 4. Identification of motor and anchor
chambers.(A) Single-molecule DNA loop extrusion
on Sytox orange (SxO)–stained surface-tethered
l-phage DNA (48.5 kbp) molecules by ATTO647N-
labeled condensin with TEV cleavage sites in kleisin
chambers I (Brn1TEV141) or II (Brn1TEV434). Starting
and end positions of the DNA loop are highlighted by
yellow and blue arrowheads, respectively. (B) The
position of condensin 0.5 to 1 s after DNA loop
rupture was scored as nondetectable (white),
back at the loop start site (yellow), or on
the translocating end of the loop (blue) (ns, not
significant; ****p <10−^12 ; Fisher’s exact test).
(C) Histogram of ATTO647N-condensin fluorescence
lifetimes at the loop start site after loop rupture.
(D) Schematic representation of the experiment
and results.
0
20
40
60
80
Loop rupture events
A
TEV
434
TEV
141
BC
DNA
Condensin
Brn1TEV141
0 s 19 s 20 s 28 s 0 s 27 s 28 s 32 s 0 s 22 s 23 s 28 s
Brn1
5 s 10 s 5 s 10 s 5 s 10 s
Loop rupture Loop rupture Loop rupture
Counts
Brn1TEV434
Fluorescence lifetime after rupture
(^00) 5 10 15 20 >20 s
10
20
30
0
5
10
15
(^20) Brn1
Counts
1 μm 1 μm 1 μm
TEV434
II
I
D
Extrude loop Cut motor
entity
Cut anchor
entity
TEV141
Brn1TEV141
ns

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