title: 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.
abstract: 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-(3)H-SP, but not methyl-(3)H-SP, with SP lyase and AdoMet results in transfer of (3)H to AdoMet, while no tritiated 5'-deoxyadenosine is observed. When 5'-tritiated AdoMet is used in the reaction with unlabeled SP, transfer of (3)H 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 beta-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.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cheek
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
mesh_terms: D004260:DNA Repair / Q000502:physiology*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D011506:Proteins; D012436:S-Adenosylmethionine / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D011506:Proteins; D012436:S-Adenosylmethionine; D006859:Hydrogen; C088014:spore photoproduct lyase; D004255:Deoxyribodipyrimidine Photo-Lyase