Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates
Nature Communications,
Год журнала:
2025,
Номер
16(1)
Опубликована: Янв. 8, 2025
Abstract
Human
DNA
licensing
initiates
replication
fork
assembly
and
replication.
This
reaction
promotes
the
loading
of
hMCM2-7
complex
on
DNA,
which
represents
core
replicative
helicase
that
unwinds
during
S-phase.
Here,
we
report
reconstitution
human
using
purified
proteins.
We
showed
in
vitro
is
specific
results
high-salt
resistant
double-hexamers.
With
ATPγS,
an
hORC1-5-hCDC6-hCDT1-hMCM2-7
(hOCCM)
assembles
independent
hORC6,
but
hORC6
enhances
double-hexamer
formation.
determined
hOCCM
structure,
hORC-hCDC6
recruits
via
five
hMCM
winged-helix
domains.
The
structure
highlights
how
hORC1
activates
hCDC6
ATPase
uncovered
unexpected
role
for
disassembly.
identified
binding
to
hORC1-5
stabilises
hORC2-DNA
interactions
supports
hMCM3-dependent
recruitment
hMCM2-7.
Finally,
allowed
us
locate
cancer-associated
mutations
at
hCDC6-hMCM3
interface,
defects.
Язык: Английский
How similar are the molecular mechanisms of yeast and metazoan genome replication initiation?
Biochemical Society Transactions,
Год журнала:
2025,
Номер
53(02)
Опубликована: Март 7, 2025
DNA
replication
start
sites
are
licensed
for
when
two
hexameric
ring-shaped
motors
of
the
replicative
helicase
loaded
as
an
inactive
double
hexamer
around
duplex
DNA.
Activation
requires
untwisting
helix
and
ejection
one
strand
from
central
channel
each
ring.
The
process
initiation
is
best
understood
in
yeast,
thanks
to
reconstitution
with
purified
yeast
proteins,
which
allowed
systematic
structural
analysis
process.
Orthologs
most
factors
have
been
identified
higher
eukaryotes;
however,
metazoan
still
its
infancy,
loading
but
not
activation
having
achieved.
Nonetheless,
artificial
intelligence-driven
structure
prediction
cryo-EM
studies
on
native
complexes,
combined
cell-based
cell-free
approaches,
starting
provide
insights
into
mechanisms.
Here,
we
describe
emerging
picture.
Язык: Английский
DNA bending mediated by ORC is essential for replication licensing in budding yeast
Proceedings of the National Academy of Sciences,
Год журнала:
2025,
Номер
122(14)
Опубликована: Апрель 4, 2025
In
eukaryotes,
the
origin
recognition
complex
(ORC)
promotes
assembly
of
minichromosome
maintenance
2
to
7
complexes
into
a
head-to-head
double
hexamer
at
DNA
in
process
known
as
replication
licensing.
this
study,
we
present
series
cryoelectron
microscopy
structures
yeast
ORC
mutants
with
DNA.
We
show
that
Orc6,
smallest
subunit
ORC,
utilizes
its
transcription
factor
II
B-B
domain
orchestrate
sequential
binding
addition,
Orc6
plays
role
scaffold
by
stabilizing
basic
patch
(BP)
Orc5
for
capture
and
bend
Importantly,
disrupting
bending
through
mutating
three
key
residues
Orc5-BP
impairs
ORC's
ability
promote
initiation
two
points
during
pre-RC
process.
This
study
dissects
multifaceted
orchestrating
activities
on
underscores
vital
Язык: Английский