Journal of the American Chemical Society
Communication
This is the second example in which the octameric histone
core catalyzes cleavage of a DNA lesion within a nucleosome.
The implications of accelerated L cleavage within nucleosomal
DNA are significant with respect to the mechanism of action of
antitumor agents such as neocarzinostatin (NCS), C1027, and
other enediyne agents.22−24 For instance, NCS produces 2-
deoxyribonolactone as part of a bistranded (“clustered”) lesion
in which the complementary strand is directly cleaved within
2−3 nucleotides of L. Although isolated abasic sites such as L
are efficiently incised during base excision repair, clustered
lesions are much less efficiently repaired.25,26 AP sites with
clustered lesions have been suggested to exist for as long as 1
day in mammalian cells.27 When considered together, these
data suggest that formation of 2-deoxyribonolactone as part of a
clustered lesion in cellular DNA results in de facto double
strand scission, the most deleterious of DNA lesions, and
provides additional chemical foundation to explain why
antitumor agents that produce it are such potent cytotoxins.
Scheme 1
migration and confirmed that 3′-phosphate groups were
exclusively formed upon L cleavage. A similar procedure
using DNA 32P-labeled at dA101 and treatment of the cleaved
DNA with shrimp alkaline phosphatase and AluI restriction
endonuclease revealed that the 3′-fragments contained 5′-
phosphates exclusively.20 The observed end groups in the DNA
fragments were consistent with β,δ-elimination of L. The
elimination mechanism was probed using dideuterated L.20
Deuterium was incorporated (94%) at C2 of the photochemical
L precursor, and nucleosome core particles containing L89 were
prepared as described above. The rate constant for disappear-
ance of the starting nucleosome was (5.3 0.3) × 10−6 s−1 (t1/2
= 36.3 h), yielding a kinetic isotope effect of 4.3, which
indicates that deprotonation is the rate-determining step.20
Previous studies of AP89 cleavage in nucleosomes showed
that the histone H4 protein is involved in ∼95% of the strand
scission reactions.10 Deleting the 19 N-terminal amino acids
reduced the rate constant ∼3-fold. The N-terminal tail of
histone H4 is rich in Lys residues, and their post-translational
modification is important in the regulation of transcription.6,7 If
they are involved in DNA lesion chemistry, their post-
translational modification could affect the stability of damaged
DNA. Consequently, we probed the role of Lys residues in the
N-terminal 20 amino acids of histone H4 on 2-deoxyribono-
lactone cleavage by preparing a series of nucleosomes
containing mutant proteins (Table 2). Replacing individual or
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for all experiments, complete
sequences of all DNAs used to prepare nucleosome core
particles, representative autoradiograms and kinetic plots, and
mass spectra of oligonucleotides containing 1 and mutated
histone H4 proteins. This material is available free of charge via
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for generous financial support from the
National Institute of General Medical Sciences (GM-063028).
We thank Ms. Hillary Clark for preparing the Lys16Ala and
Lys20Ala mutant histone H4 proteins.
Table 2. Effects of Histone H4 Mutations on 2-
Deoxyribonolactone Cleavage at SHL 1.5 (L89)
REFERENCES
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a
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a
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