RESEARCH NEWS & VIEWS

molecules surprisingly adopt a simple, cubiclike symmetry a bit like scaffolding on the
outside of a building, or, more accurately,
between two buildings.
The authors work brings together themes of
fluid behaviour near surfaces that date back to
at least the 1890s, the time of Johannes van der
Waals studies in this area. From his and related
work, we know that the pressure inside a
liquid surface is higher than the pressure outside if the liquid surface is convex, and lower
than that outside if the surface is concave3
(Fig.1). If a small droplet of liquid is confined
between two sheets, and does not wet the surfaces that is, it does not bond readily to the
surface, as is the case for mercury on glass
then the liquidvapour interface is convex and
the pressure inside the liquid must be greater
than that outside.
In fact, the pressure within a sheet-confined
droplet is determined by the ratio of the surface
tension of the liquid to the radius of curvature
of the liquids surface3. Although the precise
details of this effect at the molecular level
become complicated by atomic interactions4,5,
if the radius of curvature becomes very small
(of the order of 1nanometre or less, as in
Algara-Siller and colleagues work), then large
pressures are required to hold the liquid in
place. This is, apparently, exactly what happens
when water is sandwiched between graphene
sheets, because there are no points in the sheets
to which water can form hydrogen bonds.
How can that pressure be maintained?
To explain this, Algara-Siller et al. draw on
another theme from van der Waals theory,
namely, that all atoms must be attracted to
each other, irrespective of whether they can
form hydrogen bonds or not. This attractive
force called the van der Waals force or London dispersion force6 increases as atoms
approach each other. The authors calculate
that when sheets of atoms, such as the carbon
atoms in graphene, are separated by distances
of less than 1nm (as used in the experiment),
then the van der Waals forces can easily generate pressures as high as 1GPa.
Water trapped between graphene sheets
under these conditions is likely to crystallize, even at room temperature. But the fact
that it forms a square structure is unexpected.
The researchers computational moleculardynamics simulations do suggest that a square
lattice can form, as observed, but the detailed
origins of this strange arrangement remain
a mystery.
Are there any precedents for observing
square-like structures formed from water
molecules? Yes, there are some. Extensive
spectroscopic data and simulations of small
clusters of water molecules79 provide evidence that groups of water molecules can have
a near-cubic structure, with dangling hydrogen bonds available in principle to form a more
extensive network. And a study10 that combined molecular-dynamics simulations with

neutron-scattering experiments concluded

that the dynamics of water molecules trapped
in carbon nanotubes could be explained if the
molecules form a stationary, nearly square
array wrapped in a cylinder at the inner surface
of the nanotube, through which more water
molecules are transported in a chain-like configuration. But Algara-Siller and colleagues are
the first to directly observe an extended, twodimensional, square-like structure in water
experiments.
It remains to be seen whether the authors
observations are relevant to water transport
through naturally occurring nanometre-scale
channels. For example, aquaporin is a widely
occurring channel protein that regulates the
flow of water across the cell membrane. The
flow-control mechanism entails a combination
of hydrophobic and hydrophilic interactions
between the water and the inside surface of the
channel11, which is circular in cross-section,
not planar, as in the case of the graphene pore.
Nonetheless, the graphene results are highly
intriguing, and will probably stimulate much
debate about the nature of water in biological

channels and at surfaces. No doubt van der

Waals would have been delighted to know
that the fundamental forces that he identified so long ago have led to the discovery of an
unexpected phase of water today.
Alan K. Soper is at the ISIS Facility, Science
and Technology Facilities Council, Rutherford
Appleton Laboratory, Harwell Oxford, Didcot
OX11 0QX, UK.
e-mail: alan.soper@stfc.ac.uk
1. Petrenko, V. F. & Whitworth, R. W. Physics of Ice
(Oxford Univ. Press, 1999).
2. Algara-Siller, G. et al. Nature 519, 443445 (2015).
3. Rowlinson, J. S. & Widom, B. Molecular Theory of
Capillarity Int. Ser. Monogr. Chem. (Clarendon, 1982).
4. Lei, Y. A., Bykov, T., Yoo, S. & Zeng, X. C. J. Am. Chem.
Soc. 127, 1534615347 (2005).
5. Xue, Y.-Q., Yang, X.-C., Cui, Z.-X. & Lai, W.-P. J. Phys.
Chem. B 115, 109112 (2011).
6. London, F. Trans. Faraday Soc. 33, 8b26 (1937).
7. Gruenloh, C. J. et al. Science 276, 16781681 (1997).
8. Keutsch, F. N. & Saykally, R. J. Proc. Natl Acad. Sci.
USA 98, 1053310540 (2001).
9. Perera, P. N. et al. Proc. Natl Acad. Sci. USA 106,
1223012234 (2009).
10. Kolesnikov, A. et al. Phys. Rev. Lett. 93, 035503 (2004).
11. Agre, P. & Kozono, D. FEBS Lett. 555, 7278 (2003).

M O L EC UL A R B I OLOGY

DNA replication
reconstructed
Chromosomes must be faithfully duplicated in each cell-division cycle to ensure
genome integrity. The in vitro reconstitution of DNA-replication initiation in
yeast allows mechanistic studies of this fundamental process. See Article p.431
MICHAEL WEINREICH

n page431 of this issue, Yeeles etal.1

report the long-awaited in vitro reconstitution of the initiation of DNA replication using 16 purified proteins from the
budding yeast Saccharomyces cerevisiae. This
study defines, for the first time, the minimum
set of proteins required to initiate DNA replication in eukaryotes (organisms that include
plants, animals and fungi). The authors also
use their system to examine the regulatory
mechanism that limits initiation to just once
per cell cycle at each initiation site, confirming
and adding key details to what was previously
known.
Genome duplication must occur before cell
division, so that both progeny cells inherit a
complete copy of the genetic material. DNA
replication is initiated at particular chromosomal sites called origins, after the binding
and recruitment of several initiation proteins.
In eukaryotes, replication initiation occurs at
hundreds to thousands of origins distributed
along multiple chromosomes, with one origin

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for every 40kilobases of DNA in yeast, and

greater intervals in mammals. The activation
(firing) of these origins must be strictly regulated so that it occurs only during the DNAsynthesis period (Sphase) of each cell-division
cycle. Also, each origin must be activated once
per cell cycle at most, to avoid potentially lethal
over-replication events.
Yeeles and colleagues findings represent a
major technical feat that will allow eukaryotic
initiation to be studied in detail. The authors
work on invitro reconstitution would not have
been possible without the considerable genetic
and biochemical work of the past 30years
particularly in budding yeast, for which the
numerous proteins required for replication
initiation have been identified and the complex
regulatory processes described24. Not surprisingly, this core replication machinery has been
conserved throughout evolution, from yeast
to mammals.
The initiation of DNA replication can
be separated into two distinct and mutually exclusive steps (Fig. 1). In the first step,
which occurs in the G1 phase of the cell cycle,

NEWS & VIEWS RESEARCH

Inactive
MCM helicase

Helicase
loader

DDK + CDK

Loading
factors

d
DNA polymerase
-primase

Mcm10

Cdc45

Active
CMG helicase

GINS

G1 S
G1 phase, CDK

CDK

DDK

Figure 1 | DNA-replication initiation. a,As cells enter the G1 phase of

the cell cycle, levels of the CDK enzyme fall. This allows the helicase-loader
protein complex to recruit two hexamers of the inactive MCM helicase
enzyme complex to origin sequences throughout the genome. Several proteins
required for MCM loading are lost during this process. b,Levels of CDK and
of the DDK enzyme rise just before Sphase begins. This prevents further

an inactive form of a DNA-helicase enzyme

complex (MCM) is loaded at all replication origins throughout the genome; DNA
helicases separate and unwind the doublestranded DNA helix into two single strands.
Strand separation is necessary for the DNA to
serve as the template for its own replication,
which occurs at proteinDNA structures
called replication forks the points at which
the double-stranded DNA is unwound into
two single strands.
In the second step of initiation, the loaded
DNA helicase is activated by the recruitment
of further protein components. Helicase activation occurs locally at each active origin just
before and during Sphase. Initiation proteins
also recruit DNA primase and DNA polymerases, enzymes required to synthesize two
daughter strands on the basis of the template
sequence of the parental strands.
To prevent any single origin from firing
more than once per cell cycle, the helicase
must be loaded at the beginning of the cell
cycle (G1phase), the only period in which the
activity of the cyclin-dependent kinase (CDK)
enzyme is low. CDK belongs to the proteinkinase family of enzymes, which covalently
modify a protein or small molecule by adding a phosphate group, thereby changing the
molecules biological properties. High CDK
levels prevent further helicase loading, but are
required for helicase activation. CDK activity
thus acts as a switch between the two replication steps. A second protein kinase, DDK, is
also required for helicase activation and accumulates towards the end of G1phase.
This intricate coupling of helicase loading
and activation to protein-kinase activity is necessary to prevent re-replication of the genome5.
The activation of origins outside Sphase, or
more than once per cell cycle, would generate
amplified DNA regions that could break when
chromosomes are segregated during mitosis;
such breaking would probably be lethal. Yeeles
and co-workers study defines the minimum

MCM loading and enables loading factors to deposit two more proteins,
Cdc45 and GINS, on each of the MCM hexamers to form two active CMG
helicases. c,Yeeles etal.1 report that these CMG complexes require yet
another protein, Mcm10, to unwind origin DNA. d,DNA replication begins
after the recruitment of DNA polymerase -primase, the enzyme that initiates
DNA synthesis.

set of protein substrates for CDK and DDK

that are required for initiation invitro, and
confirms previous work5. CDK and DDK
phosphorylate many amino-acid residues in
their substrates, so researchers can now use the
authors invitro system to map which phosphorylation events are essential and how these
modifications affect proteinprotein inter
actions and biological activities.
The MCM helicase, which is composed of
six subunits, is loaded around double-stranded
DNA as a dodecamer consisting of two identical hexamers in a head-to-head arrangement6.
How this occurs is still an open question,
because the only identified intermediates7 for
this process have a single hexamer engaged
with double-stranded DNA. It will be important to determine whether the two hexamers
are loaded by two helicase loaders, sequentially
by one helicase loader, or perhaps as an intact
dodecamer8.
This work also
This major
opens up many other
technical feat
areas of investigation,
will allow
DNA-replication such as how the helicase is activated, and
initiation in
how polymerases are
eukaryotic
recruited and couorganisms to be
pled to the helicase at
studied in detail. the replication fork.
Helicase activation
requires CDK, DDK and two other proteins
to form a complex called the CMG helicase9,10.
However, Yeeles and colleagues find that CMG
cannot unwind origin DNA effectively by itself
another protein (Mcm10) is also required.
Investigating the interactions between the
helicase subunits, how each contributes to the
initial unwinding event and how the initial
dodecamer is split into two hexameric units,
are exciting research opportunities that are
now clearly possible.
Yeeles et al. observe that the DNA polymerase required for leading-strand synthesis is
recruited during the helicase activation step.

However, their in vitro system currently lacks

other components needed for chromosomal
replication. These include: the DNA polymerase that makes the lagging strand; the sliding
clamp that tethers DNA polymerases to DNA;
the clamp loader; a triad of proteins called the
fork-protection complex; and the proteins
required for the final processing steps (maturation) of the replication products. Much work
is therefore still required to reconstitute the
complete replication reaction.
If an in vitro system could be devised that
incorporates a synthesis reaction including
both leading and lagging strands, one could
imagine reconstituting the assembly of chromatin the complex of histone proteins and
DNA in the cell nucleus behind each fork, as
occurs in cells. Chromatin, rather than naked
DNA, is the true template for DNA replication
in the cell. Finally, it will be interesting to see
whether the authors system could eventually
be used to investigate mechanisms for maintaining fork stability and for replication-coupled DNA repair. These crucial processes are
clearly involved in maintaining genome integrity, and are of particular interest because they
are often lost in cancerous cells.
Michael Weinreich is in the Genome
Integrity and Tumorigenesis Laboratory,
Van Andel Institute, Grand Rapids,
Michigan 49503, USA.
e-mail: michael.weinreich@vai.org
1. Yeeles, J. T. P., Deegan, T. D., Janska, A., Early, A. &
Diffley, J. F. X. Nature 519, 431435 (2015).
2. Waga, S. & Stillman, B. Annu. Rev. Biochem. 67,
721751 (1998).
3. Heller, R. C. et al. Cell 146, 8091 (2011).
4. Li, Y. & Araki, H. Genes Cells 18, 266277 (2013).
5. Labib, K. Genes Dev. 24, 12081219 (2010).
6. Remus, D. et al. Cell 139, 719730 (2009).
7. Sun, J. et al. Nature Struct. Mol. Biol. 20, 944951
(2013).
8. Yardimci, H. & Walter, J. C. Nature Struct. Mol. Biol.
21, 2025 (2014).
9. Costa, A. et al. Nature Struct. Mol. Biol. 18, 471477
(2011).
10. Fu, Y. V. et al. Cell 146, 931941 (2011).

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