共 64 条
Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2
被引:19
作者:
Bheda, Poonam
[1
]
Swatkoski, Stephen
[2
]
Fiedler, Katherine L.
[2
]
Boeke, Jef D.
[3
,4
]
Cotter, Robert J.
[2
]
Wolberger, Cynthia
[1
,5
]
机构:
[1] Johns Hopkins Univ, Sch Med, Dept Biophys & Biophys Chem, 725 N Wolfe St, Baltimore, MD 21205 USA
[2] Johns Hopkins Univ, Sch Med, Dept Pharmacol & Mol Sci, Baltimore, MD 21205 USA
[3] Johns Hopkins Univ, Sch Med, Dept Mol Biol & Genet, Baltimore, MD 21205 USA
[4] Johns Hopkins Univ, Sch Med, High Throughput Biol Ctr, Baltimore, MD 21205 USA
[5] Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Baltimore, MD 21205 USA
来源:
基金:
美国国家科学基金会;
美国国家卫生研究院;
关键词:
SILENCING PROTEIN SIR2;
CELL-CYCLE;
TELOMERIC HETEROCHROMATIN;
SIR2-LIKE PROTEINS;
MASS-SPECTROMETER;
STRAND BREAKS;
YEAST;
DEACETYLASE;
GENE;
DNA;
D O I:
10.1073/pnas.1121471109
中图分类号:
O [数理科学和化学];
P [天文学、地球科学];
Q [生物科学];
N [自然科学总论];
学科分类号:
07 ;
0710 ;
09 ;
摘要:
Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the.-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.
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页码:E916 / E925
页数:10
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