Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: Evidence for a reversibly generated adenosyl radical intermediate

Spore photoproduct (SP) lyase, which catalyzes the direct reversal of SP (5-thyminyl-5,6-dihydrothymine) to thymine monomers, is the only identified nonphotoactivatable pyrimidine dimer lyase. Unlike DNA photolyase, SP lyase does not contain a flavin cofactor and does not require light for activation. Instead, preliminary studies point to the presence of an iron-sulfur cluster in SP lyase and the requirement for S-adenosylmethionine (AdoMet) for catalytic activity, suggesting that SP lyase belongs to the growing group of iron-sulfur cluster and AdoMet-dependent radical enzymes. Here we provide evidence for the role of AdoMet as a reversible deoxyadenosyl radical generator, which initiates repair by hydrogen atom abstraction from C-6 of SP. Reaction of 6-3H-SP, but not methyl-3H-SP, with SP lyase and AdoMet results in transfer of 3H to AdoMet, while no tritiated 5′-deoxyadenosine is observed. When 5′-tritiated AdoMet is used in the reaction with unlabeled SP, transfer of 3H into the repaired thymine monomers is observed. These results point to the reversible generation of a 5′-deoxyadenosyl radical intermediate, which reacts directly with the DNA lesion to initiate a radical-mediated β-scission. We also demonstrate that AdoMet is a catalytic cofactor that is not consumed during turnover. Together, these results support a novel radical-based mechanism for the repair of UV-induced DNA damage. Copyright © 2002 American Chemical Society.