Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily.
The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.
Buis
Jeffrey M
JM
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
Cheek
Jennifer
J
Kalliri
Efthalia
E
Broderick
Joan B
JB
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D003839:Deoxyadenosines / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015394:Molecular Structure; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D013440:Sulfides / Q000378:metabolism; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D003839:Deoxyadenosines; D007506:Iron-Sulfur Proteins; D011506:Proteins; D013440:Sulfides; C009484:5-thyminyl-5,6-dihydrothymine; C033714:5'-deoxyadenosine; D012436:S-Adenosylmethionine; D004247:DNA; D007501:Iron; C088014:spore photoproduct lyase; D004255:Deoxyribodipyrimidine Photo-Lyase; D013941:Thymine