Mechanistic studies on the repair of a novel DNA photolesion: The spore photoproduct

Article abstract of DOI:10.1021/ol9908676

(matrix presented) UV irradiation of spores results in the formation of the spore photoproduct. This novel DNA photolesion is repaired in the germinating spore in a reaction catalyzed by the spore photoproduct lyase. Model studies, using a simple bispyrim

Full text of DOI:10.1021/ol9908676

ORGANIC
LETTERS
1999
Vol. 1, No. 7
1065-1066
Mechanistic Studies on the Repair of a
Novel DNA Photolesion: The Spore
Photoproduct
Ryan A. Mehl and Tadhg P. Begley*
Department of Chemistry and Chemical Biology, Cornell UniVersity,
Ithaca, New York 14853
tpb2@cornell.edu
Received July 26, 1999
ABSTRACT
UV irradiation of spores results in the formation of the spore photoproduct. This novel DNA photolesion is repaired in the germinating spore
in a reaction catalyzed by the spore photoproduct lyase. Model studies, using a simple bispyrimidine, suggest that this repair reaction proceeds
by hydrogen abstraction from C6 of the spore photoproduct followed by â-scission of the bond linking the two pyrimidines and back hydrogen
atom transfer.
In contrast to the photochemistry of hydrated DNA, where
the cyclobutane pyrimidine photodimer is the major photo-
product, irradiation of DNA in spores results in the formation
of the spore photoproduct 2.1 One of the pathways for the
repair of this photolesion involves its conversion back to two
thymines in a reaction catalyzed by the spore photoproduct
lyase.2 This enzyme has recently been overexpressed from
Bacillus subtilis and found to contain a 2Fe-2S cluster. The
overexpressed enzyme does not catalyze the spore photo-
product repair reaction, possibly due to a requirement for
another protein or due to the instability of the cluster during
enzyme purification. However, the repair activity can be
detected in spore extract and was found to require S-adenosyl
methionine (SAM).3 This suggests that the adenosyl radical,
which can be generated by reduction of SAM by a reduced
iron sulfur cluster,4 is involved in the repair reaction and
that the cleavage of the spore photoproduct is likely to
proceed via a radical intermediate (Scheme 1). Thus,
Scheme 1. Proposed Repair Mechanism for the Spore
Photoproduct
(1) (a) For a review, see: Begley, T. P. ComprehensiVe Natural Products
Chemistry; Poulter, C. D., Ed.; Elsevier Science Ltd.: New York, 1999;
Vol. 5, pp 371-399. (b) Cadet, J.; Vigny, P. Bioorganic Photochemistry;
John Wiley & Sons: New York, 1990; Vol. 1, pp 96-99. (c) Setlow, P. J.
Bacteriol. 1992, 174, 2737.
(2) (a) Fajardo-Cavazos, P.; Salazar, C.; Nicholson, W. L. J. Bacteriol.
1993, 175, 1735. (b) Van Wang, T.-C.; Rupert, C. S. Photochem. Photobiol.
1977, 25, 123-127.
(3) Rebeil, R.; Yubo, S.; Chooback, L.; Pedraza-Reyes, M.; Kinsland,
C.; Begley, T. P.; Nicholson, W. L. J. Bacteriol. 1998, 180, 4879.
hydrogen atom abstraction from C6 of the spore photoproduct
2 by the adenosyl radical, followed by â-scission of the bond
linking the pyrimidines and back transfer of the hydrogen
10.1021/ol9908676 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/09/1999

describe our efforts to test this hypothesis using a simple
bispyrimidine model system (12).
Scheme 2. Synthesis of the Model System
We have synthesized 12, a precursor to the C6 spore
photoproduct radical, and the spore photoproduct 13 as
outlined in Scheme 2. This route involved the development
of new barbiturate reduction chemistry for the conversion
of 10 to 11, 12, and 13. Treatment of 12 under radical-
generating conditions resulted in the clean formation of
dimethylthymine 15 in 85% yield (Scheme 3). Only a trace
Scheme 3. Spore Photoproduct Fragmentation
of 13 was detected in the reaction mixture. This suggests
that the C6 radical of the spore photoproduct can undergo a
facile â-scission reaction and is a likely intermediate for the
enzymatic repair reaction.5,6
Acknowledgment. This research was supported by a
grant from the Petroleum Research Fund which is adminis-
tered by the American Chemical Society.
Supporting Information Available: Synthetic procedures
for the formation of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL9908676
(4) For a review, see: Frey, P. A. ComprehensiVe Natural Products
Chemistry; Poulter, C. D., Ed.; Elsevier Science Ltd.: New York, 1999;
Vol. 5, pp 205-223.
(5) An alternative mechanism for the cleavage reaction involving the
addition of a putative active site cysteine to C6′ of 2 followed by a retro-
Michael reaction and a formal hydride transfer is unlikely because treatment
of 13 with cysteine and base under conditions known to result in facile
H/D exchange at C5 of uracil did not result in cleavage of the spore
photoproduct. Mehl, R. A.; Nicewonger, R.; Begley, T. P. Unpublished
results.
(6) (a) Pogolotti, A. L.; Santi, D. V. Bioorganic Chemistry; van Tamelen,
E. E., Ed.; Academic Press: New York, 1977; Vol. 1, pp 277-311. (b)
Wataya, Y.; Hayatsu, H.; Kawazoe, Y. J. Biochem. 1973, 73, 871-877.
atom would complete the cleavage reaction. The energetically
unfavorable back transfer of the hydrogen atom may be
facilitated by the stabilization of the adenosyl radical by the
iron-sulfur cluster. Alternatively, the spore photoproduct
hydrogen atom transfer chemistry may be mediated by a
glycyl radical formed by hydrogen atom abstraction from
the protein by the adenosyl radical.4 In this Letter, we
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