generated in a hanging drop vapor diffusion crystallization experiment.
Diffraction data were indexed and scaled using HKL2000 (41). Molecular
replacement was carried out using Phaser (42) with the previously deter-
mined Rtt109 structure (16) and a monomer Vps75 structure (8) as search
Mass Spectrometry Quantitation Assay. Enzymatic acetylation sites were
determined using chemical acetylation combined with MS/MS peptide se-
quencing as described previously (43). The details of specific experiments
are described in SI Text.
Yeast Assays. The details of yeast strains are described in SI Text. For immu-
noblotting experiments, alkaline whole-cell extracts were prepared from
2 mL of cells grown to an OD
of 0.8 in YPD or Leu media (44). The final
volume of the extracts was 30 μL, from which 2–10 μL was loaded onto 17%
SDS-PAGE for Western blot analysis.
ACKNOWLEDGMENTS. We thank K. Luger (Colorado State University, Fort
Collins, CO) for generously supplying histone plasmids. We thank all members
in the research group of John Denu for helpful discussion s. We also acknowl-
edge the University of Wisconsin–Madison Human Proteomics Program,
funded by the Wisconsin Partnership Fund. This work was supported by
National Institutes of Health Grants GM059785 (to J.M.D.) and GM055712
(to P.D.K.) and a postdoctoral fellowship (to E.M.K.) from the American Heart
1. Shahbazian MD, Grunstein M (2007) Functions of site-specific histone acerylation and
deacetylation. Annu Rev Biochem 76:75–100.
2. Carrozza MJ, Utley RT, Workman JL, Cote J (2003) The diverse functions of histone
acetyltransferase complexes. Trends Genet 19:321–329.
3. Roth SY, Denu JM, Allis CD (2001) Histone acetyltransferases. Annu Rev Biochem
4. Brown CE, Lechner T, Howe L, Workman JL (2000) The many HATs of transcription
coactivators. Trends Biochem Sci 25:15–19.
5. Boudreault AA, et al. (2003) Yeast enhancer of polycomb defines global Esa1-
dependent acetylation of chromatin. Genes Dev 17:1415–1428.
6. Grant PA, et al. (1999) Expanded lysine acetylation specificity of Gcn5 in native
complexes. J Biol Chem 274:5895–5900.
7. Allard S, et al. (1999) NuA4, an essential transcription adaptor/histone H4 acetyl-
transferase complex containing Esa1p and the ATM-related cofactor Tra1p. EMBO J
8. Berndsen CE, et al. (2008) Molecular functions of the histone acetyltransferase
chaperone complex Rtt109-Vps75. Nat Struct Mol Biol 15:948–956.
9. Fillingham J, et al. (2008) Chaperone control of the activity and specificity of the
histone H3 acetyltransferase Rtt109. Mol Cell Biol 28:4342–4353.
10. Park YJ, Sudhoff KB, Andrews AJ, Stargell LA, Luger K (2008) Histone chaperone
specificity in Rtt109 activation . Nat Struct Mol Biol 15:957–964.
11. Tsubota T, et al. (2007) Histone H3-K56 acetylation is catalyzed by histone chaperone-
dependent complexes. Mol Cell 25:703–712.
12. da Rosa JL, Boyartchuk VL, Zhu LJ, Kaufman PD (2010) Histone acetyltransferase
Rtt109 is required for Candida albicans pathogenesis. Proc Natl Acad Sci USA
13. Driscoll R, Hudson A, Jackson SP (2007) Yeast Rtt109 promotes genome stability by
acetylating histone H3 on lysine 56. Science 315:649–652.
14. Berndsen CE, et al. (2007) Nucleosome recognition by the piccolo NuA4 histone
acetyltransferase complex. Biochemistry 46:2091–2099.
15. Xhemalce B, et al. (2007) Regulation of histone H3 lysine 56 acetylation in Schizosac-
charomyces pombe. J Biol Chem 282:15040–15047.
16. Tang Y, et al. (2008) Fungal Rtt109 histone acetyltransferase is an unexpected
structural homolog of metazoan p300/CBP. Nat Struct Mol Biol 15:738–745.
17. Han JH, Zhou H, Li ZZ, Xu RM, Zhang ZG (2007) The Rtt109-Vps75 histone acetyltrans-
ferase complex acetylates non-nucleosomal histone H3. J Biol Chem 282:14158–14164.
18. Masumoto H, Hawke D, Kobayashi R, Verreault A (2005) A role for cell-cycle-regulated
histone H3 lysine 56 acetylation in the DNA damage response. Nature 436:294–298.
19. Downs JA (2008) Histone H3 K56 acetylation, chromatin assembly, and the DNA
damage checkpoint. DNA Repair 7:2020–2024.
20. Chen CC, et al. (2008) Acetylated lysine 56 on histone H3 drives chromatin assembly
after repair and signals for the completion of repair. Cell 134:231–243.
21. Han JH, et al. (2007) Rtt109 acetylates histone H3 lysine 56 and functions in DNA
replication. Science 315:653–655.
22. Das C, Lucia MS, Hansen KC, Tyler JK (2009) CBP/p300-mediated acetylation of histone
H3 on lysine 56. Nature 459:113–U123.
23. Xie W, et al. (2009) Histone H3 lysine 56 acetylation is linked to the core transcriptional
network in human embryonic stem cells. Mol Cell 33:417–427.
24. Yuan J, Pu MT, Zhang ZG, Lou ZK (2009) Histone H3-K56 acetylation is important for
genomic stability in mammals. Cell Cycle 8:1747–1753.
25. Han JH, Zhou H, Li ZH, Xu RM, Zhang ZG (2007) Acetylation of lysine 56 of histone H3
catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity. J Biol
26. Burgess RJ, Zhou H, Han JH, Zhang ZG (2010) A role for Gcn5 in replication-coupled
nucleosome assembly. Mol Cell 37:469–480.
27. Fillingham J, et al. (2008) Chaperone control of the activity and specificity of the
histone H3 acetyltransferase Rtt109. Mol Cell Biol 28:4342–4353.
28. Tang Y, Meeth K, Jiang E, Luo C, Marmorstein R (2008) Structure of Vps75 and implica-
tions for histone chaperone function. Proc Natl Acad Sci USA 105:12206–12211.
29. Albaugh BN, Kolonko EM, Denu JM (2010) Kinetic mechanism of the Rtt109-Vps75
histone acetyltransferase-chaperone complex. Biochemistry 49:6375–6385.
30. Liu LF, et al. (2000) Mechanism of action of camptothecin. The Camptothecins:
Unfolding Their Anticancer Potential, eds JG Liehr, BC Giovanella, and CF Verschaegen
(New York Academy of Sciences, New York), Vol 922, pp 1–10.
31. Erkmann JA, Kaufman PD (2009) A negatively charged residue in place of histone
H3K56 supports chromatin assembly factor association but not genotoxic stress
resistance. DNA Repair 8:1371–1379.
32. Li Q, et al. (2009) The elongator complex interacts with PCNA and modulates transcrip-
tional silencing and sensitivity to DNA damage agents. PLoS Genet 5:e1000684.
33. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of
the nucleosome core particle at 2.8 angstrom resolution. Nature 389:251–260.
34. Banks DD, Gloss LM (2003) Equilibrium folding of the core histones: The H3-H4
tetramer is less stable than the H2A-H2B dimer. Biochemistry 42:6827–
35. Baxevanis AD, Godfrey JE, Moudrianakis EN (1991) Associative behavior of the histone
(H3-H4)2 tetramer—Dependence on ionic environment. Biochemistry 30:8817–8823.
36. Stavropoulos P, Nagy V, Blobel G, Hoelz A (2008) Molecular basis for the autoregula-
tion of the protein acetyl transferase Rtt109. Proc Natl Acad Sci USA 105:12236–12241.
37. Doyon Y, Selleck W, Lane WS, Tan S, Cote J (2004) Structural and functional conserva-
tion of the NuA4 histone acetyltransferase complex from yeast to humans. Mol Cell
38. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from
amino-acid sequence data. Anal Biochem 182:319–326.
39. Tanaka Y (2004) Expression and purification of recombinant human histones. Methods
40. Berndsen CE, Denu JM (2005) Assays for mechanistic investigations of protein/histone
acetyltransferases. Methods 36:321–331.
41. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscilla-
tion mode. Methods in Enzymology, eds CW Carter, Jr and RM Sweet (Academic, New
York), Vol 276, pp 307–326.
42. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674.
43. Smith CM (2005) Quantification of acetylation at proximal lysine residues using
isotopic labeling and tandem mass spectrometry. Methods 36:395–403 .
44. Kushnirov VV (2000) Rapid and reliable protein extraction from yeast. Yeast
20280 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1009860107 Kolonko et al.