Communications
DOI: 10.1002/anie.201005228
DNA Photochemistry
Elucidation of Spore-Photoproduct Formation by Isotope Labeling**
Gengjie Lin and Lei Li*
UV light is lethal to most microorganisms owing to its
relatively high energy and efficient absorption by biomacro-
molecules. However, bacterial endospores, which are respon-
sible for a number of human diseases, such as anthrax and
tetanus, are extremely resistant to UV radiation. For example,
some Bacillus spores can be 100 times more resistant to UV
light than the corresponding vegetative cells.[1] The highly
unusual UV resistance is due to unique DNA photochemistry
coupled with efficient damage repair in endospores.[2–8]
Scheme 1. Formation of spore photoproduct upon the exposure of
endospore DNA to UV radiation, and subsequent DNA repair by spore
photoproduct lyase.
Progress has been made in the elucidation of the damage-
repair process.[6–9] However, although SP was discovered
nearly half a century ago,[10] and despite the strong interest of
the scientific community in how it is formed,[2,11] little is
known about the mechanism of spore-DNA photochemis-
the CD3 moiety migrated to the 6-position; such a conclusion
was seriously undermined by the incomplete deuterium
incorporation observed, which led Cadet and co-workers to
conclude that the origin of the two C6 hydrogen atoms was
unclear.[12] On the basis of these results, a concerted reaction
mechanism involving the methyl group of one T residue and
try.[12]
Thymine (T) is the most UV-sensitive nucleobase.[12] In
typical cells after photochemical excitation, a T residue can
dimerize with an adjacent T residue to generate cyclobutane
pyrimidine dimers and pyrimidine (6–4) photoproducts. In
contrast, the dominant DNA photoproduct in endospores is a
unique thymine dimer, 5-thyminyl-5,6-dihydrothymine, also
called spore photoproduct or SP.[13] Spores express a specific
enzyme, spore photoproduct lyase (SPL), which effectively
reverses SP-dimer formation at the early germination phase
and thus enables spores to resume their normal life cycle
(Scheme 1).[2,13]
Although two mechanisms were proposed previously to
explain SP formation, neither of them seemed to solve the
problem unambiguously.[2,12] Varghese and Wang suggested
that the SP was formed by a consecutive mechanism through
recombination of a 5-a-thyminyl and a 5,6-dihydrothymin-5-
yl radical;[14,15] however, how these two radicals were gen-
erated was unknown. By employing [D3]thymidine containing
a CD3 moiety, Cadet and co-workers isolated the SP as a
mixture of the 5S and 5R diastereomers after the photo-
reaction. More importantly, two thirds of the generated SP
contained a deuterium atom on carbon atom C6. Although
this finding appeared to suggest that a deuterium atom from
À
the C5 C6 double bond of the other T residue was pro-
posed.[2,12]
Recently, the steric configuration of SP was determined by
using the dinucleotide thymine dinucleoside monophosphate
(TpT) instead of thymidine in a dry-film reaction.[11] The
phosphate linker and the deoxyribose groups in TpT restrict
the relative position of the Tresidues. Consequently, only one
SP TpT species, formed through the addition of the CH3
group of the 3’-T residue to the C5 atom of the 5’-T residue,
was observed by NMR spectroscopy, whereby the newly
formed C5 stereocenter adopted an R configuration.[11] The
SP TpT species generated in this manner exhibited identical
properties to those of the compound obtained by the photo-
irradiation of calf-thymus DNA, followed by enzyme diges-
tion.[6] Furthermore, only the SP with the 5R configuration,
and not the isomer (prepared by chemical synthesis) with the
5S configuration, is repaired by SPL.[16] All these results
suggest that the 5R TpT SP is truly the biologically relevant
species.
Now that the steric configuration of SP has been revealed,
the photoreaction mechanism through which it is generated
remains to be elucidated. In SP, the C6 atom of 5’-T is bonded
to two hydrogen atoms and is prochiral. As the formation of
this prochiral center constitutes a major structural change
during the photoreaction, it should be possible to shed light
on the mechanism by deciphering the origins of these
hydrogen atoms. Although we were puzzled by the observa-
tion of only 60% deuterium incorporation in the study by
Cadet and co-workers, we felt that it was a reasonable
assumption that one H atom is retained from the original 5’-T
residue, whereas the other is derived from the methyl group
of 3’-T during the photochemical formation of SP. To test this
hypothesis, we prepared two deuterium-labeled TpT dinu-
[*] Dr. G. Lin, Prof. Dr. L. Li
Department of Chemistry and Chemical Biology
Indiana University-Purdue University Indianapolis (IUPUI)
402 North Blackford Street, Indianapolis, IN 46202 (USA)
Fax: (+1)317-274-4701
E-mail: lilei@iupui.edu
[**] We thank Prof. Eric Long at IUPUI for helpful discussions, and the
National Institute of Environmental Health Sciences
(R00ES017177) as well as the IUPUI startup fund for financial
support. The NMR and MS facilities are supported by National
Science Foundation MRI grants CHE-0619254 and DBI-0821661,
respectively.
Supporting information for this article is available on the WWW
9926
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 9926 –9929