A str
ategy w
as developed to
assemble nucleosomes specific
ally d
am
aged
at only one site
andone structur
al orient
ation. The most prev
alent UV photoproduct,
a cis-syn cyclobut
ane thymine dimer (
csCTD), w
as chemic
ally synthesized
and incorpor
ated into
a 30 b
ase oligonucleotide h
arboring theglucocorticoid hormone response element. This oligonucleotide w
as
assembled into
a 165 b
ase p
air doublestr
anded DNA molecule with nucleosome positioning elements on e
ach side of the
cs CTD-cont
aininginsert. Proton NMR verified th
at the synthetic photoproduct is the
cis-syn stereoisomer of the CTD.Moreover, two different pyrimidine dimer-specific endonucle
ases cut ~90% of the dsDNA molecules.This cle
av
age is completely reversed by photore
activ
ation with
E. coli UV photoly
ase, further demonstr
atingthe correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone oct
amerexch
ange from chicken erythocyte core p
articles,
and cont
ained
a unique tr
ansl
ation
al
and rot
ation
al settingof the insert on the histone surf
ace. Hydroxyl r
adic
al footprinting demonstr
ates th
at the minor groove
atthe
cs CTD is positioned
aw
ay from the histone surf
ace
about 5 b
ases from the nucleosome dy
ad.Competitive gel-shift
an
alysis indic
ates there is
a sm
all incre
ase in histone binding energy required forthe d
am
aged fr
agment (
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G ~ 0.15 kc
al/mol), which does not prevent complete nucleosome lo
adingunder our conditions. Fin
ally, folding of the synthetic DNA into nucleosomes dr
am
atic
ally inhibits cle
av
age
at the
cs CTD by T4 endonucle
ase V
and photorevers
al by UV photoly
ase. Thus, specific
ally d
am
agednucleosomes c
an be experiment
ally designed for in vitro DNA rep
air studies.