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 liquid–vapour 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 liquid’s 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 1 nanometre 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 1 nm (as used in the experiment), then the van der Waals forces can easily generate pressures as high as 1 GPa. 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 molecules7–9 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: [email protected] 1. Petrenko, V. F. & Whitworth, R. W. Physics of Ice (Oxford Univ. Press, 1999). 2. Algara-Siller, G. et al. Nature 519, 443–445 (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, 15346–15347 (2005). 5. Xue, Y.-Q., Yang, X.-C., Cui, Z.-X. & Lai, W.-P. J. Phys. Chem. B 115, 109–112 (2011). 6. London, F. Trans. Faraday Soc. 33, 8b–26 (1937). 7. Gruenloh, C. J. et al. Science 276, 1678–1681 (1997). 8. Keutsch, F. N. & Saykally, R. J. Proc. Natl Acad. Sci. USA 98, 10533–10540 (2001). 9. Perera, P. N. et al. Proc. Natl Acad. Sci. USA 106, 12230–12234 (2009). 10. Kolesnikov, A. et al. Phys. Rev. Lett. 93, 035503 (2004). 11. Agre, P. & Kozono, D. FEBS Lett. 555, 72–78 (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
O
n page 431 of this issue, Yeeles et al.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 40 kilobases 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 (S phase) 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 in vitro reconstitution would not have been possible without the considerable genetic and biochemical work of the past 30 years — particularly in budding yeast, for which the numerous proteins required for replication initiation have been identified and the complex regulatory processes described2–4. 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
a
Inactive MCM helicase
Helicase loader
b
c
DDK + CDK
Loading factors
d DNA polymerase α-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 S phase 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 protein–DNA 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 S phase. 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 (G1 phase), 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 molecule’s 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 G1 phase. 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 S phase, 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 et al.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 α-primase, the enzyme that initiates DNA synthesis.
set of protein substrates for CDK and DDK that are required for initiation in vitro, and confirms previous work5. CDK and DDK phosphorylate many amino-acid residues in their substrates, so researchers can now use the authors’ in vitro system to map which phosphorylation events are essential and how these modifications affect protein–protein 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: [email protected] 1. Yeeles, J. T. P., Deegan, T. D., Janska, A., Early, A. & Diffley, J. F. X. Nature 519, 431–435 (2015). 2. Waga, S. & Stillman, B. Annu. Rev. Biochem. 67, 721–751 (1998). 3. Heller, R. C. et al. Cell 146, 80–91 (2011). 4. Li, Y. & Araki, H. Genes Cells 18, 266–277 (2013). 5. Labib, K. Genes Dev. 24, 1208–1219 (2010). 6. Remus, D. et al. Cell 139, 719–730 (2009). 7. Sun, J. et al. Nature Struct. Mol. Biol. 20, 944–951 (2013). 8. Yardimci, H. & Walter, J. C. Nature Struct. Mol. Biol. 21, 20–25 (2014). 9. Costa, A. et al. Nature Struct. Mol. Biol. 18, 471–477 (2011). 10. Fu, Y. V. et al. Cell 146, 931–941 (2011).
2 6 M A RC H 2 0 1 5 | VO L 5 1 9 | NAT U R E | 4 1 9
© 2015 Macmillan Publishers Limited. All rights reserved
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: [email protected] 1. Petrenko, V. F. & Whitworth, R. W. Physics of Ice (Oxford Univ. Press, 1999). 2. Algara-Siller, G. et al. Nature 519, 443–445 (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, 15346–15347 (2005). 5. Xue, Y.-Q., Yang, X.-C., Cui, Z.-X. & Lai, W.-P. J. Phys. Chem. B 115, 109–112 (2011). 6. London, F. Trans. Faraday Soc. 33, 8b–26 (1937). 7. Gruenloh, C. J. et al. Science 276, 1678–1681 (1997). 8. Keutsch, F. N. & Saykally, R. J. Proc. Natl Acad. Sci. USA 98, 10533–10540 (2001). 9. Perera, P. N. et al. Proc. Natl Acad. Sci. USA 106, 12230–12234 (2009). 10. Kolesnikov, A. et al. Phys. Rev. Lett. 93, 035503 (2004). 11. Agre, P. & Kozono, D. FEBS Lett. 555, 72–78 (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
O
n page 431 of this issue, Yeeles et al.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
4 1 8 | NAT U R E | VO L 5 1 9 | 2 6 M A RC H 2 0 1 5
© 2015 Macmillan Publishers Limited. All rights reserved
for every 40 kilobases 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 (S phase) 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 in vitro reconstitution would not have been possible without the considerable genetic and biochemical work of the past 30 years — particularly in budding yeast, for which the numerous proteins required for replication initiation have been identified and the complex regulatory processes described2–4. 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
a
Inactive MCM helicase
Helicase loader
b
c
DDK + CDK
Loading factors
d DNA polymerase α-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 S phase 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 protein–DNA 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 S phase. 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 (G1 phase), 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 molecule’s 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 G1 phase. 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 S phase, 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 et al.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 α-primase, the enzyme that initiates DNA synthesis.
set of protein substrates for CDK and DDK that are required for initiation in vitro, and confirms previous work5. CDK and DDK phosphorylate many amino-acid residues in their substrates, so researchers can now use the authors’ in vitro system to map which phosphorylation events are essential and how these modifications affect protein–protein 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: [email protected] 1. Yeeles, J. T. P., Deegan, T. D., Janska, A., Early, A. & Diffley, J. F. X. Nature 519, 431–435 (2015). 2. Waga, S. & Stillman, B. Annu. Rev. Biochem. 67, 721–751 (1998). 3. Heller, R. C. et al. Cell 146, 80–91 (2011). 4. Li, Y. & Araki, H. Genes Cells 18, 266–277 (2013). 5. Labib, K. Genes Dev. 24, 1208–1219 (2010). 6. Remus, D. et al. Cell 139, 719–730 (2009). 7. Sun, J. et al. Nature Struct. Mol. Biol. 20, 944–951 (2013). 8. Yardimci, H. & Walter, J. C. Nature Struct. Mol. Biol. 21, 20–25 (2014). 9. Costa, A. et al. Nature Struct. Mol. Biol. 18, 471–477 (2011). 10. Fu, Y. V. et al. Cell 146, 931–941 (2011).
2 6 M A RC H 2 0 1 5 | VO L 5 1 9 | NAT U R E | 4 1 9
© 2015 Macmillan Publishers Limited. All rights reserved
