ArticleNonenzymatic biotinylation of histone H2A |
| Shannon Healy 1 *, Tom D. Heightman 2, Laura Hohmann 3, David Schriemer 1, Roy A. Gravel 1 |
1Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1
2Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom
3Institute for Systems Biology, Seattle, Washington
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| email: Shannon Healy (shealy@ucalgary.ca) |
*Correspondence to Shannon Healy, Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1
Funded by:
The Canadian Institute for Health Research (CIHR), The CIHR Group in Medical Genetics, The CIHR Strategic Training Program in Genetics, Child Development and Health at the University of Calgary
| biotin histones nonenzymatic holocarboxylase synthetase mass spectrometry |
Holocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio-5 -AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15-23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio-5-AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928-1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N-terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation. |
Received: 25 September 2008; Revised: 30 October 2008; Accepted: 4 November 2008
10.1002/pro.37
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