inaccessible to bulk methods and relevant to
many interactions between proteins and ligands.
The use of an inherently nonequilibrium method to obtain equilibrium binding energies also
grants access to molecular interactions that equilibrate over very long time scales (e.g., nucleosome assembly) and that can only be currently
measured by indirect techniques such as competition assays (2, 22, 34). The FTLB relates work
measurements to binding energies without making any assumption on reaction kinetics or the
ideal solution limit. Therefore, it might be also
used to test the explicit breakdown of the law of
mass action in conditions where it is not applicable, for instance, in crowded environments, where
ligands exhibit compartmentalized dynamics due
to steric hindrance interactions (35). Lastly, the
applicability of the FTLB is not restricted to biomolecular reactions and might be directly applied
to other interacting systems that can only be explored through nonequilibrium methods.
REFERENCES AND NOTES
1. G. D. Stormo, Y. Zhao, Nat. Rev. Genet. 11, 751–760
2. S. Leavitt, E. Freire, Curr. Opin. Struct. Biol. 11, 560–566
3. J. M. McDonnell, Curr. Opin. Chem. Biol. 5, 572–577
4. C. G. Kalodimos et al., Science 305, 386–389 (2004).
5. S. Kim et al., Science 339, 816–819 (2013).
6. C. Bustamante, Z. Bryant, S. B. Smith, Nature 421, 423–427
7. J. P. Junker, K. Hell, M. Schlierf, W. Neupert, M. Rief, Biophys. J.
89, L46–L48 (2005).
8. Y. Cao, M. M. Balamurali, D. Sharma, H. Li, Proc. Natl. Acad.
Sci. U.S.A. 104, 15677–15681 (2007).
9. D. Koirala et al., Nat. Chem. 3, 782–787 (2011).
10. S. R. K. Ainavarapu, L. Li, C. L. Badilla, J. M. Fernandez,
Biophys. J. 89, 3337–3344 (2005).
11. E. Hann et al., Biophys. J. 92, L79–L81 (2007).
12. C. Jarzynski, Phys. Rev. Lett. 78, 2690–2693 (1997).
13. G. E. Crooks, Phys. Rev. E 61, 2361–2366 (2000).
14. J. Liphardt, S. Dumont, S. B. Smith, I. Tinoco Jr., C. Bustamante,
Science 296, 1832–1835 (2002).
15. D. Collin et al., Nature 437, 231–234 (2005).
16. E. A. Shank, C. Cecconi, J. W. Dill, S. Marqusee, C. Bustamante,
Nature 465, 637–640 (2010).
17. P. Maragakis, M. Spichty, M. Karplus, J. Phys. Chem. B 112,
18. I. Junier, A. Mossa, M. Manosas, F. Ritort, Phys. Rev. Lett. 102,
19. A. Alemany, A. Mossa, I. Junier, F. Ritort, Nat. Phys. 8,
20. Materials and methods are available as supplementary materials.
21. A. Orte et al., Proc. Natl. Acad. Sci. U.S.A. 105, 14424–14429
22. B. Fierz, S. Kilic, A. R. Hieb, K. Luger, T. W. Muir, J. Am. Chem.
Soc. 134, 19548–19551 (2012).
23. H. Yu et al., Proc. Natl. Acad. Sci. U.S.A. 109, 5283–5288
24. D. R. Lesser, M. R. Kurpiewski, L. Jen-Jacobson, Science 250,
25. L. Jen-Jacobson, Biopolymers 44, 153–180 (1997).
26. B. J. Terry, W. E. Jack, R. A. Rubin, P. Modrich, J. Biol. Chem.
258, 9820–9825 (1983).
27. S. J. Koch, A. Shundrovsky, B. C. Jantzen, M. D. Wang, Biophys.
J. 83, 1098–1105 (2002).
28. J. SantaLucia Jr., Proc. Natl. Acad. Sci. U.S.A. 95, 1460–1465
29. J. M. Huguet et al., Proc. Natl. Acad. Sci. U.S.A. 107,
30. M. M. Van Dyke, P. B. Dervan, Science 225, 1122–1127
31. C. Bailly, F. Hamy, M. J. Waring, Biochemistry 35, 1150–1161
32. F. Leng, J. B. Chaires, M. J. Waring, Nucleic Acids Res. 31,
33. P. O. Heidarsson et al., Proc. Natl. Acad. Sci. U.S.A. 111,
34. A. Thåström, J. M. Gottesfeld, K. Luger, J. Widom, Biochemistry
43, 736–741 (2004).
35. S. Schnell, T. E. Turner, Prog. Biophys. Mol. Biol. 85, 235–260
We acknowledge funding from the European Research Council (ERC)
MagReps (grant 267 862), Framework Programme 7 Infernos
(grant 308850), the Catalan Institution for Research and Advanced
Studies (ICREA) Academia 2013, and Ministerio de Economia y
Competitividad (grant FIS2013-47796-P). All data used in this study
are included in the main text and in the supplementary materials.
Materials and Methods
Figs. S1 to S10
Tables S1 to S16
4 July 2016; accepted 16 December 2016
RPA binds histone H3-H4 and
functions in DNA replication–coupled
Shaofeng Liu,1,2 Zhiyun Xu,1 He Leng,1,3 Pu Zheng,1 Jiayi Yang,1 Kaifu Chen,4
Jianxun Feng,1 Qing Li1†
DNA replication–coupled nucleosome assembly is essential to maintain genome integrity
and retain epigenetic information. Multiple involved histone chaperones have been
identified, but how nucleosome assembly is coupled to DNA replication remains elusive.
Here we show that replication protein A (RPA), an essential replisome component that
binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA
directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound
single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex
formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in
H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we
propose that RPA functions as a platform for targeting histone deposition to replication fork,
through which RPA couples nucleosome assembly with ongoing DNA replication.
Nucleosome assembly during S phase is tight- ly coupled to DNA replication (1). The initial step of replication-coupled (RC) nucleo- some assembly is the deposition of histone H3-H4 onto replicating DNA, which is fol-
lowed by the rapid deposition of histone H2A-
H2B (2, 3). Deposition of new histone H3-H4
requires the action of histone chaperones (4).
Replication protein A (RPA), a complex that in
yeast is composed of the Rfa1, Rfa2, and Rfa3
subunits, binds single-stranded DNA (ssDNA)
at replication forks after double-stranded DNA
(dsDNA) is unwound by the replicative helicase
minichromosome maintenance (MCM), facilitates
the movement of the replisome, and functions
as a “unique harbor and binding platform” during
DNA transactions (5–8). We studied the potential
roles of RPA in RC nucleosome assembly.
We first analyzed whether RPA interacts with
histone chaperones involved in RC nucleosome
assembly, including chromatin assembly factor–1
(CAF-1) (9), anti-silencing function 1 (Asf1) (10),
regulator of Ty1 transposition 106 (Rtt106) (11),
and facilitates chromatin transactions (FACT)
(12, 13). RPA subunit Rfa2 bound CAF-1, FACT,
and Rtt106, but did not bind Asf1 (Fig. 1A and fig.
S1A). CAF-1 is recruited to DNA replication forks,
in part, through its interaction with proliferating cell nuclear antigen (PCNA) (14–16). The
RPA–CAF-1 interaction was unaffected in PCNA
mutant (pol30-879) cells defective for the PCNA–
CAF-1 interaction (16) (Fig. 1B), indicating that
the RPA–CAF-1 interaction occurs independently of the PCNA–CAF-1 interaction. Moreover, histone H3-H4 promoted the interaction between
RPA with each histone chaperone in vitro (fig.
S1, B to E).
RPA copurifies with both histones H3 and H4
(17). We confirmed this interaction and showed
that the RPA–H3-H4 interaction was not mediated by DNA (Fig. 1C). In vitro binding assays
demonstrated that recombinant RPA directly
binds free H3-H4, but not (mono)nucleosomal
1State Key Laboratory of Protein and Plant Gene Research,
School of Life Sciences and Peking-Tsinghua Center for Life
Sciences, Peking University, Beijing 100871, China. 2Peking
University–Tsinghua University–National Institute of
Biological Sciences Joint Graduate Program, School of Life
Sciences, Tsinghua University, Beijing 100084, China.
3Academy for Advanced Interdisciplinary Studies, Peking
University, Beijing 100871, China. 4Center for Cardiovascular
Regeneration, DeBakey Heart and Vascular Center, Houston
Methodist, Houston, TX 77030, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: firstname.lastname@example.org