Lewin Benjamin (ed.) Genes IX

ili.i;3lf ]lS-li

MicrococcaI nuctease

digests chromatin in

nuclei into

a

multimeric

series of DNA

bands that can be

separated by

gel

etectrophoresis. Photo

courtesy of

Markus

Nott,

Urniversitdt Ziirich.

DNA

Is

Coited

in Arrays

of

Nucleosomes

>95%

of the

DNA is recovered

in nucleosomes or

muttimers when micrococcaI nuctease

cteaves

DNA

of chromatin.

The Length of DNA

per

nucteosome varies for

individual, tissues

jn

a range from 154

to

260

bp.

When chromatin is digested

with the enzyme

micrococcal nuclease, the DNA

is cleaved

into

integral multiples of a

unit

length. Fractiona-

tion by

gel

electrophoresis reveals the

"ladder"

presented

in

Fitil"jfi*

Jt3"f .

Such

ladders

extend

for

-I0

steps, and the unit length.

determined

by

the increments between successive steos. is

-200

bp.

i:i.r1ifii

i!r.it shows that the ladder is

gener-

ated by

groups

of

nucleosomes.

When

nucleo-

somes are fractionated on a sucrose

gradient,

they

give

a series of discrete

peaks

that corre-

spond to monomers, dimers, trimers, and so on.

When the DNA

is

extracted from the individ-

ual fractions and electrophoresed,

each

fraction

yields

a

band

of DNA

whose size corresponds

with

a step on the

micrococcal

nuclease ladder.

The monomeric nucleosome

contains

DNA of

the unit length, the nucleosome dimer contains

DNA

of

twice the unit length, and

so

on.

Each step on the ladder represents the DNA

derived

from a discrete number of nucleosomes.

We therefore take the existence of the 200 bp

ladder

in any chromatin

to indicate that the DNA is orga-

il?i.rll[1I

;ti].;i

Each multimer

of

nucteosomes

contains

the

appropriate

number of unit

[engths

of DNA.

In the

photo,

artjficiaI bands simulate

a

DNA ladder.

The

image was con-

structed using

PCR fragments

with sizes

corresponding

to

actuaI band sizes.

Photo courtesy

ofJan

Kieteczawa,

Wyeth

Research.

nized into nucleosomes

The micrococcal

ladder

is

generated

when

only

-2o/o

of

the

DNA in the

nucleus is rendered

acid-soluble

(degraded to

small

fragments)

by the

enzyme.

Thus

a small

proportion

of

the DNA

is specifically

attacked;itmust

represent

especially

susceptible

regions.

When chromatin

is spilled

out

of nuclei,

we

often

see a series

of

nucleosomes

connected

by

a thread of

free

DNA

(the

beads

on a string).

The need

for tight

packaging of

DNA in

vivo,

however, suggests

that

probably there

is usually

little

(if

any)

free

DNA.

Extract

DNA and

electrophorese

c

z

o

P

rooo

o

J

800

600

400

29.3

DNA Is Coiled

jn

Arravs of

Nuc[eosomes

767

This

view is confirmed

by the fact that>95yo

0f the DNA

of chromatin can

be recovered in the

form

of the

200 bp ladder. Almost

all DNA must

there-

fore

be organized in nucleosomes.

In

their nat-

ural

state, nucleosomes

are

likely

to be closely

packed,

with DNA

passing

directly from

one to

the

next. Free DNA is

probably

generated

by

the loss

of some histone

octamers during

isolation.

The length

of DNA

present

in the nucleo-

some varies

somewhat from

the

"typical"

value

of 200

bp. The chromatin

of any

particular

cell

type has a

characteristic average

value

(+5

bp).

The

average

most often is

between 180 and 200,

but there

are extremes

as

low

as 154

bp

(in

a

fungus)

or as high

as 260 bp

(in

a sea urchin

sperm). The

average value may

be different in

individual

tissues

of the adult organism,

and

there

can be differences

between

different

parts

of the

genome

in a

single cell type.

Variations

from

the

genome

average include

tandemly

repeated

sequences,

such as clusters

of 5S RNA

senes.

@

Nucteosomes

Have

a Common

Structure

r

Nucleosomal

DNA is divided into

the

core

DNA

ano

tinker DNA

depending

on

its

susceptibiLity

to

micrococcaI

nuc[ease.

r

The

core DNA is

the [ength

of

146

bp that is found

on

the core

particles

produced

by

prolonged

digestion with micrococcaI

nuclease.

.

Linker DNA

is the region

of 8 to 71,4

bp that is

susceptibte

to early

cleavage by the

enzyme.

o

Changes

in the

length of [inker DNA

account for

the variation

jn

total

length of nucleosomal

DNA.

r

H1

is associated

with linker DNA

and may

lie at

the

point

where

DNA enters

and leaves

the

nucteos0me.

A

common

structure

underlies

the varying

amount

of DNA that is

contained

in nucleo-

somes of

different

sources. The

association

of

DNA

with the histone

octamer forms

a core

par-

ticle containing

146

bp

of

DNA,

irrespective

of

the total

length

of DNA in

the nucleosome.

The

variation

in

total length

of DNA

per

nucleo-

some is

superimposed

on this

basic core

structure.

The

core

particle

is defined

by the

effects of

micrococcal

nuclease

on the nucleosome

monomer.

The initial reaction

of the

enzyme is

to cut

between nucleosomes,

but if it

is allowed

to

continue

after monomers

have

been

sener-

CHAPTER

29

Nucteosomes

Base

parrs

180

160

140

Time

of dioestion

-.>

tgliilftl

t:1.*

Micrococcal

nuclease reduces

the

lenqth

of nucteosome

monomers in

discrete steps. Photo

cour-

tesy of Roger Kornberg.

Stanford University

School of

Medicine.

ated, then it

proceeds

to digest

some of the DNA

of the individual

nucleosome.

This occurs

by a

reaction

in which DNA is

"trimmed"

from the

ends

of the nucleosome.

The

length of the DNA

is reduced

in dis-

crete steps, as shown in

!:ttjljftf

;i*.t.

With

rat

liver

nuclei, the nucleosome

monomers

initially

have 205

bp of DNA. After the first

step.

some

monomers

are found in

which

the length

of

DNA has

been reduced to

-I65

bp. Finally,

this

is reduced

to the length

of the DNA

of the core

particle,

146bp.

(The

core is reasonably

stable,

but

continued digestion

generates

a

"limit

digest." In the limit

digest, the longest

fragments

are the 146

bp DNA

of the core,

whereas

the

shortest are

as small as 20 bp.)

This

analysis suggests

that the nucleosomal

DNA can

be divided into

two regions:

o

Core DNA has

an

invariant

length

of

146 bp, and is relatively

resistant

to

digestion by nucleases.

o

Linker

DNA comprises

the rest

of the

repeating

unit. Its

length

varies from

as

little

as 8 bp

to as much

as 114

bp

per

nucleosome.

The

sharp size

of the band

of DNA

gener-

ated by the initial

cleavage with

micrococcal

nuclease

suggests

that the region

immediately

available

to

the enzyme is restricted.

It

represents

only

part

of each linker.

(If

the entire linker

DNA

were

susceptible,

the band

would range

from 146 bp to

>200

bp.) Once

a cut has been

made in

the iinker DNA,

though, the rest

of this

region becomes

susceptible,

and it can be

removed relatively

rapidly

by further

enzyme

action. The connection

between nucleosomes

is represented in iili":,i-ilill ,a;i.'i

ii.

Core

particles

have

properties

similar to

those of the

nucleosomes

themselves, although

they are smaller. Their

shape and

size are sim-

ilar

to those of nucleosomes;

this suggests that

the essential

geometry

of the

particle

is

estab-

lished

by the

interactions

between DNA and

the

protein

octamer

in the core

particle.

Core

particles

are more readily

obtained as a homo-

geneous population,

and as a result they are

often used for structural

studies in

preference

to nucleosome

preparations. (Nucleosomes

tend

to vary because it is difficult

to obtain a

prepa-

ration in

which there has

been no end-

trimming of the DNA.)

What

is

the

physical

nature

of the core and

the

linker regions?

These terms

were introduced as

operational definitions that

describe the regions in

terms of their relative

susceptibility to nuclease

treat-

ment.This description does not

make any impli-

cation

about

their actual structure. It

turns out,

though, that the major

part

of the core DNA is

tightly curved on the nucleosome,

whereas the

terminal regions

of the core and the linker

regions are

more

extended

(see

Section 29.5,

DNA

Structure Varies on the

Nucleosomal

Surface).

The existence

of

linker

DNA depends on

factors

extraneous to the four

core

histones.

Reconstitution experimenls

in

vitro

show that

histones have an intrinsic

ability to organize

DNA into core

particles,

but do not form nucie-

osomes with the

proper

unit

length.

The degree

of supercoiling of the DNA is an important fac-

tor. Histone Hl and/or nonhistone

proteins

influence the length

of

linker

DNA associated

with the

histone

octamer in a natural

series ol

nucleosomes.

"Assembly

proteins"

that are not

part

of the nucleosome structure

are

involved

in vivo in constructing nucleosomes

from his-

tones and DNA

(see

Section

29.9,

Reproduc-

tion of Chromatin

Reouires

Assemblv of

Nucleosomes).

Where

is histone Hl located?

The Hl is

lost during the degradation of nucleosome

monomers.

It

can be retained on monomers

that still have 165 bp of DNA, but is always lost

with the

final reduction

to the 146 bp core

par-

ticle. This suggests that HI could be located in

the

region

of the

linker DNA immediately

adja-

cent to the core DNA.

MicrococcaI

nuctease

initiatlv

cleaves

between

nucte-

osomes. Mononucteosomes

typicatty

have

-200

bp DNA.

End-

trimming reduces the

Length of

DNA first to

-165

bp, and then

gen-

erates core

particles

with 146 bp.

If Hl

is located at the

linker,

it

could

"seal"

the DNA in the

nucleosome

by binding

at the

point

where

the

nucleic acid enters

and

leaves

(see

Figure

29.4).The

idea that

Hl lies in the

region

joining

adjacent

nucleosomes

is consis-

tent with old

results that

Hl is

removed the

most readily from

chromatin,

and that

Hl-

depleted

chromatin

is more

readily

"solubi-

lized." In

addition,

it is easier

to obtain

a

stretched-out

fiber of beads

on a string

when

the

Hl has

been

removed.

DNA Structure

Varies on

the

NucleosomaI

Surface

o

DNA is wrapped

1.65 times

around the

histone

octamer.

o

The structure of the

DNA is altered

so that

it has

an

jncreased

number of base

pairs/turn

in the

middte. but a decreased

number at the

ends.

The exposure of

DNA on

the surface

of the

nucleosome explains

why

it is accessible

to

cleavage by certain

nucleases.

The reaction

with

nucleases that attack

single

strands

has been

especially

informative.

The enzymes

DNAase

I

and DNAase II

make single-strand

nicks in

DNA;

they cleave

a bond

in one strand,

but

the other

strand

remains

intact at this

point. Thus no effect

is

visible

in the double-stranded

DNA. Upon

denaturation,

though,

short

fragments

are

released instead of

full-length

single

strands.

If

the DNA

has been

labeled at

its ends,

the end

fragments

can be

identified

by autoradiogra-

phy,

as summarized

in

r

lilijltf,

i:ii.t

:.

When

DNA

is free in solution,

it is nicked

(relatively)

at

ran-

dom.

The DNA on

nucleosomes

also can

be

nicked by the

enzymes,

but

only at

regular

intervals.

When

the

points

of

cutting

are

+

200bp

*

165bp

+

146bp

Mono-

Trimmed

Core

particles

nucleosomes

29.5

DNA Structure

Varies

on the

Nucleosoma[

Surface

763

Electrophoresis

Labeled fragment

Labeled

fragment+

i::+ijiiF

l:*..1 1

Njcks in

double-stranded DNA

are

revealed

by

fragments

when the DNA is

denatured

to

give

singte strands.

If the DNA is labeled at

(say)

5'ends. on[y the 5'fragments are visible

by

autoradiography. The

size of the fragment identifies

the distance of the

nick

from the [abeled

end.

irii;t-:*.ir

;-::"i: Sites for nicking

lie at regutar interva[s

atong core

DNA, as

seen

in

a DNAase I

digest of nuclei.

Photo

courtesy

of Leonard

C.

Lutter,

Henry Ford

Hospita[,

Detroit,

ML

determined

by using radioactively

end-labeled

DNA

and

then DNA is

denatured

and electro-

phoresed,

a Iadder

of the

sort displayed in

i:ii.;F:a

l:ij..1: is

obtained.

The interval

between successive

steps

on

the ladder

is l0

to I L bases. The

ladder

extends

for the full

distance

of core DNA.

The cleavage

sites

are numbered

as

Sl through

Sl3

(where

CHAPTER

29

Nucteosomes

Sl is

-I0

bases from

the

labeled

5'end,

52 is

-20

bases

from it,

and so on). Their

positions

relative

to the DNA superhelix

are illustrated

in

F{*i.iR[:*"13.

Not all sites are cut with equal

frequency:

Some are cut rather

efficiently, whereas

others

are

cut scarcely at all. The enzymes

DNAase I and

DNAase

II

generate

the

same

ladder,

although

with some differences in the intensities

of the

bands. This shows

that the

pattern

of cutting

represents

a unique series of

targets in DNA.

determined by its organization,

with only

some

slight

preference

for

particular

sites imposed

by

the individual

enzyme. The same

cutting

pat-

tern is

obtained by cleaving

with a hydroxyl

radical,

which argues that

the

pattern

reflects

the

structure

of the

DNA

itself rather

than any

sequence

preference.

The

sensitivity of nucleosomal

DNA

to

nucleases is

analogous to a footprinting

exper-

iment. Thus

we can assign the lack

of reaction

at

particular

target sites to the

structure

of the

nucleosome,

in

which certain

positions

on DNA

are rendered

inaccessible.

There

are two strands

of DNA in

the core

particle,

so in an end-labeling

experiment

both

of

the 5'

(or

3') ends are labeled,

one on each

strand. Thus

the cutting

pattern

includes

frag-

ments

derived from

both strands.

This is implied

in Figure 29.I1,

where

each labeled

fragment

is derived from

a different

strand. The

corollary

is that, in

an experiment,

each labeled

band in

fact

can

represent

two fragments

that are

gen-

erated

by cutting lhe

same distance

hotli. either

of the labeled

ends.

s12

s11

s10

S9

S8

s7

S6

JC

764

How, then, should

we interpret

discrete

preferences

at

particular

sites? One

view

is

that

the

path

of DNA

on the

particle

is

symmetrical

(about

a horizontal axis

through the nucleo-

some,

as drawn in Figure 29.4).

If., for exam-

ple,

no S0-base fragment

is

generated

by

DNAase I, this must

mean that

the

position

at

80 bases from the 5'end

of. either strand is not

susceptible to the

enzyme. The

second

num-

bering scheme used in Figure 29.13

reflects this

view, and identifies

57

=

site 0 as the center

of

symmetry.

When

DNA is

immobilized

on a flat sur-

face, sites are cut

with a regular separation.

ijltii.ii:l: ;.:i:r.

lii

suggests that this reflects

the

recur-

rence of the exposed

site with the helical

peri-

odicity of B-form DNA. The

cutting

periodicity

(the

spacing between

cleavage

points)

coincides

with-indeed, is

a

reflection

of-the

structural

periodicity (the

number

of base

pairs per

turn

of the double helix). Thus

the distance between

the

sites

corresponds

to the number

of

base

pairs

per

turn.

Measurements

of

this

type suggest

that the average value for double-helical B-type

DNA is 10.5 bp/turn.

What

is

the

nature

of the target

sites on the

nucleosome? riir\i+ii-:

;i'.i.:i r: shows that each site

has three to four

positions

at which cutting

occurs; that is, the

cutting site is defined t2 bp.

Thus

a cutting site

represents

a short

stretch of

bonds on both strands that is exposed

to

nucle-

ase action over three to four

base

pairs.

The rel-

ative

intensities indicate

that some sites are

preferred

to others.

From this

pattern,

we can calculate the

"average"

point

that is cut. At the ends of the

DNA,

pairs

of sites from Sl

to 54 or

from

Sl0

to S

I

3

lie

apart

a

distance of 10.0 bases each. In

the center of the

particle,

the

separation

from

sites 54 to S

I 0 is I

0.7 bases.

(This

analysis deals

with average

positions,

so sites need not lie

at

an integral number of

bases apail.)

The

variation

in

cutting

periodicity

along

the core DNA

(10.0

at the ends, 10.7 in

the

mid-

dle) means that there is variation in the struc-

tural

periodicity

of core DNA. The DNA has

more bp/turn than its solution value in the mid-

dle, but has fewer bp/turn at the ends. The aver-

age

periodicity

over

the

nucleosome is

only

10.17 bp/turn, which is significantly less than

the

10.5 bp/turn of DNA in

solution.

The crystal structure of the core

particle

suggests that

DNA is

organized as a flat super-

helix, with 1.65 turns wound

around

the his-

tone octamer.

The

pitch

of the superhelix varies

and

has

a discontinuity

in

the middle.

Regions

Side

view

Top

view

i'li:tii:il- 'l:i, i

:; Two numbering

schemes divide

core

particle

DNA

into 1.0

bp segments.

Sites

may be

numbered S1 to 513

from one

end; or taking 57 to

identify coordinate

0 of

the dyad symmetry,

thev mav be

numbered

-7

to

+7.

ii.ii:iiili: :i:i;j,

i n

The most exposed

positions

on DNA

recur

with a

periodicity

that

reflects the structure

of the dou-

bte hetix.

(For

ctarity,

sites are shown

for

onty

one strand.)

;iIi:i,ii-li:

;:i;,, !

-,

High

resotution anatysis

shows

that each

site

for DNAase I consists

of severaI

adjacent

susceptibte

phosphodiester

bonds,

as seen

in this

exampte of

sites

54 and S5 anatyzed

in end-tabeted

core

particles.

Photo courtesy

of Leonard

C.

Lutter.

Henry Ford

Hospitat.

Detroit, MI.

29.5

DNA Structure

Varies

on the

Nucteosomal

Surface

765

@

The

Periodicity

of

DNA

Changes

on the

Nuc[eosome

o -0.6

negative

turns of DNA

are absorbed

by the

change in

bp/turn from 10.5 in

solutjon

to an

average

of 10.2 on the nucteosomaI

surface. which

explains

the [inking-number

paradox.

Some

insights into

the structure

of nucleoso-

mal DNA

emerge

when we

compare

predic-

tions

for supercoiling

in

the

path

that DNA

follows

with actual measurements

of supercoil-

ing

of nucleosomal

DNA.

Much

work on the

structure

of sets of nucleosomes

has

been car-

ried

out with

the virus SV40.

The DNA

of SV40

is a

circular molecule

of 5200

bp, with

a con-

tour length

-1500

nm.

In both

the virion

and

infected

nucleus,

it is

packaged

into

a series of

nucleosomes,

which

together

are called

a

minichromosome.

As

usually isolated,

the

contour length

of the

minichromosome

is

-2

l0 nm,

which

corre-

sponds

to a

packing

ratio

of

^-7

(essentially

the

same

as the

-6

of

the nucleosome

itself).

Changes

in the

salt concentration

can

convert

it

to a flexible

string

o{ beads

with a much lower

overall

packing

ratio.

This

emphasizes

the

point

CHAPTER

29 Nucteosomes

that nucleosome strings can take more

than one

f.orminvitro, depending on

the conditions.

The degree

of supercoiling on the indi-

vidual nucleosomes

of the minichromosome

can be

measured

as illustrated in

ft{ilJft*

!l$.11 *.

First, the free supercoils

of the minichro-

mosome itself

are

relaxed,

so that

the nu-

cleosomes form

a circular string

with a

superhelical density of 0.

Next, the histone

octamers are extracted. This releases

the DNA

to

follow

a free

path.

Every

supercoil that

was

present

but constrained in the minichromo-

some wili appear in the

deproteinized DNA

as

-l

turn.

Now

the

total

number

of supercoils

in the SV40 DNA is measured.

The

observed

value

is close

to the number

of nucleosomes. The reverse result

is seen

when

nucleosomes

are assembledinvitro

onto a super-

coiled SV40 DNA: The formation

of each nucle-

osome removes

-l

negative

supercoil.

Thus the DNA follows

a

path

on the nucle-

osomal surface that

generates

-l

negative

super-

coiled turn

when the

restraining protein

is

removed.

The

path

that DNA

follows

on the

nucleosome,

though, corresponds

to

-1.67

superhelical

turns

(see

Figure 29.4).

This dis-

crepancy is sometimes called

the linking num-

ber

paradox.

of high curvature

are arranged symmetrically,

and occur

at

positions

+l

and

+4.

These

corre-

spond

to 56 and S8, and

to 53 and SI l, respec-

tively,

which are the

sites

least

sensitive to

DNAase

I.

A high-resolution

structure

of the nucleo-

some

core shows in detail how

the structure of

DNA is

distorted. Most of the

supercoiling occurs

in the

central 129 bp, which

are coiled into 1.59

left-handed

superhelical turns

with a diameter

of

80

A

(only

four

times the diameter

of the

DNA duplex itself

).

The terminal

sequences on

either

end make

only a very small contribution

to

the overall

curvature.

The central 129

bp are in

the

form

of B-

DNA.

but

with a substantial

curvature that is

needed

to form the

superhelix. The major

groove

is smoothly

bent, but the minor

groove

has

abrupt kinks.

These conformational

changes

may explain

why the

central

part

of nucleoso-

mal DNA

is not

usually a target for

binding by

reguiatory proteins,

which typically

bind to the

terminal

parts

of the core

DNA or to

the

linker

sequences.

I

Remove

proteins

i

Supercoiled DNA

I

Treat

with topoisomerase

FJ{itJ}Jfl

tg".'r{:: The

supercoils

of the SV40 mjnichromo-

some

can be retaxed

to

generate

a cjrcutar

structure,

whose

loss

of hjstones

then

generates

supercoi[s

in the free

DNA.

L9L

rauepo auolsrH aqlJo

uoqezluefuo

L'62

Jdeqs

Jql suroJ

lI

'Jarxepo

eqt

Jo

JJlJruprp

eql

roJ slunoJJE J3ruprlat

ZVH-T€H

aqJ

'/

I

'62

eJn

-3lg

ul

,{.llerrteruaq)s

pelerlsnilr

se,r,t

teqt

eAIl

-radsrad

erues aql sluasardar.lzran dol aq1

'seuolsIq

IenpIAIpuI

Moqs

tou

seop

tnq

(an1q)

sraurp

gZH-VZH

aql

uror;

(arqu)

rJuprlet

lVH-z

€Haqt

saqsrn8ultstp

Iaporu

rql snqJ

'gzH

qllM

vzH

pue

'tH

qll1l{

gg

:rauged

stl

qllM patelfrpratur

sr

qJeJ

teql

1nq

'suralord

relnqop

lenprlrpu

se

pazrueS:o

lou

JJp sauolsrq Jql

leqr

slsaSSns eJnlJnrls

Ipl

-s,{.n

aql uI seuoq>lJeQ apltdad,ilod

lenprntput

aqt

yo

sqted aqt SuoerJ

'.iit'i

il :irt:{ii,i:,1

uI

uMoqs

Jerrrel)o Juolsrq eql JoJ

Iepou

aql

slsaSSns

(uorlnlosar

y

I'S

le)

eJnlf,nns

1e1sLn

aq1

'tsur>IUIISSOJJ

ol asuodsar

pue

JoIAeqJq uollJeJalul

JIJq]

Jo

srspq Jql uo JJnlJnrls JrrJruelJo aql;o

suor8ar

o1

pau8rsse

uaJq enpq sJuolslq

IpnpI^IpuI

Jqt

Jo

suortrsod aq1

'JJntJnJts

lerauaS

Jq1

qsll

-qPlSJ

SUOrlJerJlUr auolsrr{-Juorsrq

eql

]eql

s1sa33ns srql

'allrupd

aror aqt

Jo

tpql

ot

JelI

-rurs

sr JJurPlJo Juolsrq

pJlPlosr

aqt

;o

adeqs

IIPJa^o

aql

lPql

l!\or{s sJrpnls

IeJnl)nJls

',:

i.'i!r:

.+llU$i.-l

uI uIIoI

rtleruruer8elp

ur uMoqs

sl

1I

'JruosoelJnu

Jql

1o

uotleztueSro

Jql JoJ

pelJnJlsuoJ

ueaq spq

lJpotu

e

'Plep

JSJqt ruor{

('suorperalur

roleru aqt riyrddt

sllnsJr

aril JJqlaqM Surpnap ur snorlneJ

aq o1

,(ressa

-Jau

sr

ll

os

'pJ>lurTSsoJJ

sJruo)Jq sulatord

aqt;o

uoruodord

fierus

e

,(1uo Llensn

teql

sr e]ep

qJns

qrpn,ltlnrryyrp

y)

'aruosoapnu

eql uI

rrqlo

qf,Pe

reJu

JII sJuotsrq

yo

srred

qJIqM .troqs o1 sdtqs

-uorleleJ

eseql

puelxJ

sJrpnls

3ur>1ur1ssor3

'apured

eJot

Jqt

Jo

sJlua

-dord

aqt;o aruos

,{.e1dsrp

teqt

saprued olur

lJit^

/ll

VNO

azrue8ro ueJ JJruerlel eql

'reuellJl

'qlPeulapun

st

raqlo

aql

lMet^ dol eql ur u00s 0q uel raurp

BZH-vzH

eu0

'edeqs

aq1

ro1

1aure1

e sepL'rord .lourp.llal

zrH-zEH

aql

'eulos

-oolrnu

oql lo1

lopour leruleuufs

e u1

: ];ii'iil]i:l

zVH-|€Haql;o

SupsrsuoJ

,,lauJJ>1,,

IerlueJ

P seq

eurosoJlJnu

Jql

qJIqM

ul uollezlueSro;o

urrol

e roy sanSre

srql'Jeluert

al

epll-z

g11

aq1 seneel

qJIqM

'1urod

slql

le

[lateredas

lsol

sI

Jeulp

flzH-vzH

raqlo aqJ'rsrulp

szH-YZH

ue

lsol

seq

teql

sauotslr1

Jo

JJIupxJq

e aleraua8

01 JIeIJ

-ossrp

ueJ JJureDo

aqJ

'uonprluaJuo)

uralord

-q8rq

pue

rles-q8tq

Jo

suolllpuoJ

rJpun 04a

zr ater)ossp

seuotslq

3u11a1

Lq

(fuprqyp

aroru

qfl^azr)

ro ullPruoJq)

ruoJJ uollJPrlxa

zlq rJI{]lJ

paurplqo

eq

ueJ sJauretJo

auolslV

1le1ul

'(AZH-VZH)

reulp

e relnrrged

ur

'g1Hpueyzll

^dq

paurro;

are saxaldruoJ

snolre1

'(lVtl-r*t)

Jeruprlel

p

uroJ

7H

pup

911

'saxald

-ruoJ

Jo

sadrh

o^4,r1 tuJoJ

seuolslq

eJoJ JqJ

'eluosoalJnu

Jql

qtlm

sluJrulradxa

SutlullssoJ)

.{q

pue

'saxald

-uo)

alqets

ruJoJ

01 sJIlITIqe

rraqt

,(q

paprrrord

sr suorlJeJJlur

Juolslq-Juolslq

lnoqe

eJuapIAJ

Jq1

Jo

tsow

ZsJeruet)o

IUJoJ

01

,{.l11qe

luapuad

-Jpur

ue ssassod,{aqr

op Jo

'YN(

1o

aruasard

eqt

ur Lluo

.{.padord

trear

sJuotslq

oC

'VNo

qll^\

pue

Jeqlo

qJea

qll^\

Deralul

sauolslq aql

.&|Oq

Mou>l 01

pJau

a,u

'ura1oJd

yo

an4radsrad

eql

ruoJd

'Jf,eJJns

Jql

uo

panueSro

sI

vN(

Jql

Moq

Jo

arruradsrad

aql uorJ

elrrosoJlJnu

Jql

Jo

uorl)nrlsuoJ

aql

pJrJpISuo)

e^eq

31!\ JeJ

snqJ

'eulosoaltnu

aql

J0

lno

puolxe sltP]

lPutullal-N

'ploJ

euolstq

aql

J0

Jtlolll

lPlnpnlls

aql a^eq

sauolstq

elol

llv

.

'lau1lPo

sl!

ql$ palelrbrpralut filanLsualxa

st auo]sttl

Lllel

r

'seurp

8zH-vzH

oMl

qlm

palPtlossP

lauPllol

zrH-zEH

ue

Jo laural

e seq raurello

auo]stt{

aLll

r

Jauello

auolstH

aql

Jo

uoqezruP6r0

'1tor-radns

e se

pernseau

aq

ol

Uel

sI

lsJJ

eqt.{po

1urn17dq

Jo

Jaqlunu

Jql SuISParJuI

olur

sao8

VNq

leurosoJl)nu

uI

uleJls

IPUoIS

-rot

aqt

Jo

euos

'DaJJa

uI

'srunl

leJlleq.radns

6'1-

Jo

tuaruJrnspau

eql

pue

49'I-

Jo

qted prtsdqd

Jqt

ueJ,lrtJq,{tuedanstp

aql

sureldxa

qJILIM

'suJnl

49'0-

sqJosqe

JruosoJlJnu

Jq1 uo

vN(I

punoM,{pq31r

ssrl aqt

lo

qled

rqJ'surnl0'6I

=

S'0I/002

sPq

Mou

1l

'euosoelJnu

eql

IUOJJ

pasPrlar

sl

VNO

ueq^A'surnl

49'6I

=

LI' 0I l00e

Jrp arJqt

'dq

OOZ

Jo

eluosoepnu

e uI

'YNQ

eJrI

Jo

urnlTdq

E'0 I

aqt

pue

VN(

IeIuosoJIJnu

1o

urnlTdq

JBpJeAe

ZI'0I

eql

uJemleq

aJue

-rJJIIp

aqt ,(q

paureldxa sr

druedaDslp

JqI

-xalJ

P

seq osle

Juotsrq

qJeE

'JtuosoalJnu

Jql

Jo

ssPlu

urJtoJd

IeJluJJ

Jql o1 sJlnqrJtuo)

]eq]

^poq

JelnqolS

p

seq sJuolsrq

JJof, Jql

Jo

q)pg

.JlnSrJ

Jql

Jo

ued

JJMOI

Jql ur

ueJS eq ueJ

se

'sllunqns

€H

oMl Jql

uaJMlJq

suoIlJeJJluI

^q

paruloJ

sr JJurerlat

|VH-|{.H

aq1

'(aruanbas;o

a,rrnadsJrJr

VN(

Lue a8eped

ol

pJJu

Jqt

qtrM

tuJlsrsuo))

sJuoq>lJeq

ralsJrp

-oqdsoqd

aqt

or Lltsoru

sr

Surpurg

('paterrsnllr

sr

]eq]

JJuJJeJrrrnJJrJ

Jql JoJ

€+

-

S'0+

1e

spurq

tH-€H

:9+

-

S'€+

le

spurq

AZH-yZH)

'xrrrrl

rlq

-nop

vN(I

eqt

Jo

sulnl

s'z

spulq

rrrulporelJq

qJee

lsraurrpoJJlJq

padeqs-luarserJ

ruJoJ ol

lJeJatur

suor8ar

JSJqJ'pIoJ

auolsrq

aql

pJIIeJ

sl slql :sdoo1

o,r,r1

Lq

panauuoJ

eJe sJf,rlJq

n

JJrql qJIqM

ur ernlJnJls

yo

ad,{1 Jplrurrs

e

Moqs

'Mar^

uolloq

aql ur sa^leq

qloq

;o

uoLlLsoduuadns

eq1 ut

uaas aq uer

uorlezLuebro

leru

-1aLuufis

eq] iauosoallnu

Jleq

e ur srLed

g7g-y711

pue

79

-tH

Moqs nneLn

do1

e uL suorlLsod

auolsrH

.

soruosoallnN

6z

ulldvHl

sJuolsrq

Jrol rnoJ

IIv

'(Z+

ot

0

uroJJ

paraqrunu)

aurosoJlJnu

Jql

punoJe

uJnl Juo 01

JAIIeJJJ

ad.,{r

qrBa

Jo

Juotsrq Juo

Jo

uorlrsod

Jqt suor{s

aaatn faddn

JQI

':,

r

':rr

rJ*r'-:iri

uI

pJZIJeuIuInS

SI

(y

s'z

re

JrnrJnrls

1elsln

e uo

paseq)

sauotsrrl

Jql

Jo

suortrsod

aqt

Jo

,trerl

pJlretep

aJotu

V

.MEIA

epls

aql

q8norql

relnlpuadrad

unr

p1no,r,r

1eq1

srxe

ue

tnoqB

L"rtauru.{s p1o;o.,r,rt

s.{eldsrp

Iepotu

eqJ

'JruosoalJnu

e

ur suJnl

IInJ

oMl

lsolule

ur

punou

rq

plnou

qtlr{M

'VNO

roJ

alrs Surpurq

Jql

ot

puodsarJo)

plnoJ

teqt

qted

IeJrTJqJedns

e

qtrar

'1oods

Jo

uos

p

suroJ uratord

aql'paqsrn8

-unsrp

Jq ueJ sJJturp

SZH-V7H

aleredas

aql

Jo

pue

JJruprlJt

zVH-.€H

Jqt

Jo

sJrlrlrqrsuodsar

Jql eJJH

'y'67

etn?r.g ur

pJtprlsnllr

seM

teql

arrttradsrad

aups Jql

sluasardar Mar^

Jprs JqJ

',r,t.Jrl

srql ur UJJS

Jq up) auo Lluo

lnq

'sJaurp

o.Ml se u1

trl

srred

fJzH-yzH

JqJ

'JoqsJsroq

p

Jo

'filLsranrul

surldop

suqo['sr)pueupno1ir1

'N

'3

1o

fiselnor

soloqd

'alpunq

aprm

-V

0Z

P u! sautl

lelleled

aq1

[q

unnoqs st Mat^

oprs aql ur

pire

'(VtrtC

Jo

relauprp

eq1 talenb auo)

aqnl

Moilpu

e sp

maLn

do1 aql ur uMoqs

s!

VN6

aql

1o

qled

lequalod

aq1

'qlpaulapun

uapprq sr leqlo

aq] asnelaq

'nneLn

do1 aq1 ut

alqrsr^ sr

slaurp

BZH-VZH

oqlJo auo r{1ug'enlq

ur uMoqs

s.rautp

BZH-VZH

oql

pue

alrqM ut u/v\oqs leuelJal

zrH-ztH

aql

qllM

lapoLu

6uq1g-ereds

e uL

paluasetdet

sr

lauelto

a.lol auolsrq

aql

Jo

ainllnlls

1e1s&t

eql

I !:

891

VH EH

BZH VZH

't

1*

uollezrueblo

;eculeutuAs

smor1s sJted

euolstq

eql

1o

uorlrsoduuedng

,,euosoalcnu

lleqr

e uilol

slred euoistH

FI#ilf{il J#"f* The 10 nm

fiberin

partiatty

unwound

state

can be seen to consist

of a string

of

nucteosomes.

Photo

courtesy of

Barbara Hamkato.

University

of Catifornja,

Irvine.

FI*i.!fiil iit"ti The

gtobutar

bodies of the histones are

locatized

in

the

histone

octamer of the core oarticte.

The

locations ofthe

N-terminal

taits, which carry the sites for

modification,

are

not

known, though, and coutd be more

ftexi bte.

'tn

f,:*tifril

3*.I1 The N-terminaI histone

taits

are disor-

dered and exit

from

the

nucleosome

between turns of

the DNA.

ible N-terminal tail, which

has

sites

for modifi-

cation

that may be important in chromatin

func-

tion.

The

positions

of the tails, which account

for about one

quarter

of the

protein

mass, are

not

so well

defined, as indicated in

Ff

tltiftS

f $.f

.

The

tails

of both Hl and H2B, however. can be

seen to

pass

between the turns of the DNA

superhelix and extend out of the

nucleosome,

as shown

in

ill*u$i* ;l*.f 1.

When histone tails

are crosslinked to

DNA

by UV

irradiation, more

products

are obtained with nucleosomes com-

pared

to core

particles,

which could mean that

the tails contact

the linker DNA. The tail of H4

appears to contact

anH2A-H2B dimer in an

adjacent

nucleosome; this could be

an impor-

tant feature

in

the overall structure.

The Path

of

Nucleosomes

the

Chromatin

Fiber

10 nm chromatin

fibers are

unfotded

from 30

nm

fibers and consist

of a string

of

nucleosomes.

30

nm fibers have

six nucteosomes/turn,

which

are

organized

into a sotenoid.

Histone

H1 is required

for formation

of

the 30

nm

fiber.

When chromatin

is examined

in the

electron

microscope, two

types

of fibers

are seen:

the

l0 nm fiber

and f 0

nm

fiber. They

are described

by

the approximate

diameter

of the thread

(that

of the 30

nm

fiber actually

varies

from

-25-30

nm).

The

l0 nm fiber

is essentially

a continu-

ous string of

nucleosomes.

In fact,

at times

it

runs

continuously

into

a

more stretched-out

region

in

which

nucleosomes

are

seen

as a string

of beads, as

indicated

in the

example

of

F5*ljtril

t+.;i;]. The

I0 nm

fibril structure

is

obtained

under

conditions

of

low

ionic strength

and does

not

require

the

presence of histone

HI. This

means that

it is

a function

strictly

of the

nucleosomes

themselves.

It may

be visualized

essentially

as a continuous

series

of nucleo-

somes, as shown

in

flI{i{JF't

il'S.!.$. It

is not clear

whether

such

a structure

exists

invivo or

is sim-

ply

a consequence

of

unfolding

during

extrac-

tion

in vitro

When

chromatin

is

visualized

in conditions

of

greater ionic strength,

the

30 nm

fiber

is

29.8

The Path

of Nucleosomes

in the

Chromatin

Fiber 769

iji,l;iti

i;":i.:::::

The

10 nm fiber is

a continuous

strinq of

nuc[eosomes.

i:iiiiii:i .il.f

+

The

30 nm fiber

has

a coited

structure.

Photo

courtesy

of Barbara

Hamka[0.

University

of Catifor-

nia,

Irvine.

;l'J',ir"s;

*T:l?T

;t',""i':i

ff :Li;,il;

coiled

structure.

It has

-6

nucleosomes

for

every

turn,

which

corresponds

to

a

packing

ratio

of

40

(that

is,

each

pm

along

the axis

of the fiber

con-

tains

40

pm

of DNA).

The

presence

of Hl is

required.

This

fiber is

the basic

constituenr

CHAPTER

29 Nucteosomes

i*l*tJF.[

i]$"il:i The 30 nm fiber is

a heticaI

ribbon con-

sisting

of two

parattel

rows

of

nucteosomes

coited

jnto

a

solenoid.

of both interphase

chromatin

and mitotic

chromosomes.

The most likely

arrangement

for

packing

nucleosomes

into

the fiber is

a solenoid,

in

which the nucleosomes

turn in a helical

array

that is

coiled around

a central

cavity. The

two

main

forrns

of a solenoid are

a single-start, which

forms

from a single

linear array,

and a two-stan,

which in effect

consists of a

double row

of nucle-

osomes. eI*{.:ff*

;S.il$

shows a two-start

model

suggested by recent

crosslinking

data identify-

ing a double

stack of nucleosomes

in the

30 nm

fiber.

This is supported

by

the crystal

structure

of a tetranucleosome

complex.

The

30 nm

and l0 nm

fibers

can be

reversibly

converted

by changing

the ionic

strength.

This

suggests that

the linear

array

of

nucleosomes

in

the l0 nm

fiber is

coiled

into

the 30 nm

structure

at higher

ionic

strength

and in the

presence

of Hl.

Although

the

presence

of Hl is necessary

for

the formation

of the l0 nm fiber,

information

about its

location is

conflicting.

Its relative

ease

of

extraction from

chromatin

seems

to

argue

that it is

present

on the outside

of

the superhe-

lical

fiber axis.

Diffraction

dara,

though,

and

the

fact

that it is

harder

to find in

30 nm fibers

than

in l0

nm fibers

that retain

it,

would

argue for

an interior

location.

770

Lewin Benjamin (ed.) Genes IX

Подождите немного. Документ загружается.